Cytoplasmic Binding Research Articles

Specific Eph receptor-cytoplasmic effector signaling mediated by SAM-SAM domain interactions.

The Eph receptor tyrosine kinase (RTK) family is the largest subfamily of RTKs playing critical roles in many developmental processes such as tissue patterning, neurogenesis and neuronal circuit formation, angiogenesis, etc. How the 14 Eph proteins, via their highly similar cytoplasmic domains, can transmit diverse and sometimes opposite cellular signals upon engaging ephrins is a major unresolved question. Here, we systematically investigated the bindings of each SAM domain of Eph receptors to the SAM domains from SHIP2 and Odin, and uncover a highly specific SAM-SAM interaction-mediated cytoplasmic Eph-effector binding pattern. Comparative X-ray crystallographic studies of several SAM-SAM heterodimer complexes, together with biochemical and cell biology experiments, not only revealed the exquisite specificity code governing Eph/effector interactions but also allowed us to identify SAMD5 as a new Eph binding partner. Finally, these Eph/effector SAM heterodimer structures can explain many Eph SAM mutations identified in patients suffering from cancers and other diseases.

CRABP1 and 2 differentially regulate HCV infection via modulation of lipid droplet abundance.

Abstract Vitamin A is an essential nutritional constituent that governs a broad spectrum of cell biology through the activation of its nuclear receptor complexes. The active metabolite of vitamin A, all-trans retinoic acid (ATRA), binds to the chaperone molecule, cytoplasmic retinoic acid binding protein (CRABP) 1 or 2 to regulate its signaling pathways. Given the extremely high homology, CRABP1 and 2 have been predicted to play a redundant role. However, a few studies by others suggested that CRABP1 and 2 might differentially regulate the cellular response to ATRA. Although the antiviral effect of ATRA is well recognized, the roles of CRABPs in the regulation of viral pathogens have never been studied. In this study, we employed hepatitis C virus (HCV) as a study model to assess the importance of CRABPs. Of great interest, CRABP1 expression enhanced HCV infection while CRABP2 expression resulted in the suppression of HCV lifecycle. Our follow up studies collectively indicated that CRABPs regulate the efficiency of HCV infection through the modulation of lipid droplet (LD) abundance wherein HCV establishes the replicase complex. The transcriptome analysis revealed that CRABP1 expression downregulated the genes regulated by ATRA and is associated with the expression of genes involved in the steatosis of the liver. In contrast, CRABP2 expression augmented ATRA response, resulting in the inhibition of LD accumulation. Moreover, the chemical inhibition of the CRABP1 and 2 pathways resulted in the abrogation of their pro- and antiviral effect, respectively. In conclusion, our study revealed a novel link between the cellular response to the micronutrients and its influence on the viral infectious disease.

Dynarrestin, a Novel Inhibitor of Cytoplasmic Dynein

Aberrant hedgehog (Hh) signaling contributes to the pathogenesis of multiple cancers. Available inhibitors target Smoothened (Smo), which can acquire mutations causing drug resistance. Thus, compounds that inhibit Hh signaling downstream of Smo are urgently needed. We identified dynarrestin, a novel inhibitor of cytoplasmic dyneins 1 and 2. Dynarrestin acts reversibly to inhibit cytoplasmic dynein 1-dependent microtubule binding and motility invitro without affecting ATP hydrolysis. It rapidly and reversibly inhibits endosome movement in living cells and perturbs mitosis byinducing spindle misorientation and pseudoprometaphase delay. Dynarrestin reversibly inhibitscytoplasmic dynein 2-dependent intraflagellar transport (IFT) of the cargo IFT88 and flux ofSmo within cilia without interfering with ciliogenesis and suppresses Hh-dependent proliferation of neuronal precursors and tumor cells. Assuch, dynarrestin is a valuable tool for probing cytoplasmic dynein-dependent cellular processes and a promising compound for medicinal chemistryprograms aimed at development of anti-cancerdrugs.

Decreased cytoplasmic X-box binding protein-1 expression is associated with poor prognosis and overall survival in patients with oral squamous cell carcinoma

IntroductionSquamous cell carcinoma is the most common cancer of the oral cavity. In spite of advancements in surgical, chemoradiological and targeted therapies, these therapeutic strategies still have had little impact on survival rates. X-box binding protein-1 (XBP-1) is a potent transcription factor that is involved in the unfolded protein response (UPR) pathway, which itself is activated in response to endoplasmic reticulum stress as a method to restore cellular homeostasis. The role XBP-1 plays in oral squamous cell carcinoma (OSCC) has yet to be determined. In this study, we used molecular and immunohistochemical analyses to investigate the role of XBP-1 protein playing in the OSCC carcinogenesis. Materials and methodsWe used immunohistochemical analyses to investigate XBP-1 expression in 255 OSCC tissue specimens, as well as migration and invasion assays with XBP-1 siRNA transfection of oral cancer cell lines to confirm its role in OSCC. ResultsThe XBP-1 immunostaining was dichotomized as low-level expression and high-level expression. We found that low-level cytoplasmic XBP-1expression was significantly correlated with larger tumor size (p=0.047), more advanced clinical stage (p<0.0001), lymph node metastasis (p=0.002), and shorter overall survival (p=0.011). Kaplan-Meier survival curves showed that low-level cytoplasmic XBP-1 expression was significantly correlated with shorter overall survival (p=0.031). The univariate Cox regression analysis revealed that cytoplasmic XBP-1 expression was a prognostic factor for overall survival of patients with OSCC. We also found that inhibition of XBP-1 promoted OSCC cell migration and invasion. ConclusionOur results suggest that XBP-1 expression may play an essential role in the pathogenesis of OSCC and that targeting XBP-1 may be a sound therapeutic strategy.

Dissecting the Forces that Dominate Dimerization of the Nucleotide Binding Domains of ABCB1

P-glycoprotein, also known as multidrug resistance protein 1 or ABCB1, can export a wide range of chemically unrelated compounds, including chemotherapeutic drugs. ABCB1 consists of two transmembrane domains that form the substrate binding and translocation domain, and of two cytoplasmic nucleotide binding domains (NBDs) that energize substrate transport by ATP binding and hydrolysis. ATP binding triggers dimerization of the NBDs, which switches the transporter from an inward facing to an outward facing transmembrane domain conformation. We performed MD simulations to study the dynamic behavior of the NBD dimer in the presence or absence of nucleotides. In the apo configuration, the NBDs were overall attractive to each other as shown in the potential of mean force profile, but the energy well was shallow and broad. In contrast, a sharp and deep energy minimum (∼−42 kJ/mol) was found in the presence of ATP, leading to a well-defined conformation. Motif interaction network analyses revealed that ATP stabilizes the NBD dimer by serving as the central hub for interdomain connections. Simulations showed that forces promoting dimerization are multilayered, dominated by electrostatic interactions between the nucleotide and conserved amino acids of the signature sequence and the Walker A motif. In addition, direct and water-bridged hydrogen bonds between NBDs provided conformation-defining interactions. Importantly, we characterized a largely unrecognized but essential contribution from hydrophobic interactions between the adenine moiety of the nucleotides and a hydrophobic surface of the X-loop to the stabilization of the nucleotide-bound NBD dimer. These hydrophobic interactions lead to a sharp energy minimum, thereby conformationally restricting the nucleotide-bound state.

Human Gastro-Intestinal Graft-Versus-Host Disease Is Mediated By Retinoic Acid-Responsive CD8+ Effector T-Cells Under IL-23 Polarising Conditions

Background: Gastro-intestinal (GI) graft-versus-host disease (GvHD) results in significant morbidity and mortality after allogeneic hematopoietic stem cell transplantation (AHSCT). Retinoic acid (RA), a metabolite of vitamin A, has diverse effects on immune cells. RA signaling promotes immune tolerance under steady state conditions. However in pro-inflammatory conditions RA increases Th1, Th17 CD4+ and Tc1 CD8+ T cell responses and is implicated in the pathogenesis of autoimmune inflammatory bowel disease. Furthermore, RA exposure and RA-receptor alpha (RARα) signaling in allogeneic T cells potentiates GI-GvHD in mice. Thus RA and cytokines that influence the effect of RA on T cells may represent potential therapeutic targets for GI-GvHD. However, the impact of RA at a tissue level in human GI-GvHD is unknown. We therefore investigated the role of RA-responsive T cells in human GvHD in vivo and human allogeneic T-cell responses in vitro .Methods: We scrutinized GI biopsies from 47 patients after AHSCT (36 with and 11 without GI-GvHD confirmed by conventional histological criteria). We also analysed skin biopsies from 11 AHSCT patients. We used conventional immunohistochemistry and a novel deep phenotyping method using sequential staining, stripping and re-probing on the same fixed embedded biopsy to determine cellular co-expression of multiple markers to identify RA-responsive T cells in human GvHD biopsies at sites of GvHD damage. Finally, we used an HLA-mismatched allogeneic mixed lymphocyte co-culture (MLRs) system, combining CFSE dye dilution and multi-parameter flow cytometry to enable the phenotyping of alloproliferative T cells after manipulation of RA signalling.Results: Firstly we observed thatthe number of cells expressing high levels of cytoplasmic RA binding proteins I/II (a surrogate marker of RA) was increased in gut biopsies in patients with GI-GvHD versus controls (median 155 vs 43 cells/mm2, p=0.056). Importantly the number of cells expressing RARα was also significantly increased (median 1315 vs 544 cells/mm2, p=0.001) consistent with RA-responsiveness. Crucially the number of RARα+ cells correlated with clinical stage of acute GI-GvHD with higher numbers in patients with severe (stage 3-4) versus less severe (stage 1-2) disease.Having demonstrated that RA-responsive cells were associated with both occurrence and severity of GI-GvHD we went on to identify potential RA-responsive T cell subsets. Additionally, as IL-23 and IL-33 direct RA-programming of T cells to either pro-inflammatory or tolerogenic phenotypes we also measured cellular expression of these cytokines and their receptors. CD8+ T cells co-localized with RARα+ cells and were present in significantly increased numbers in gut biopsies from patients with GI-GvHD, as were Tbet+ Th1/Tc1 cells. In contrast, numbers of RORγ+ Th17 cells were unchanged and CD4+ cells were decreased. IL-23p19+ and IL-23R+ cell numbers were significantly increased in GI-GvHD biopsies, whereas IL33R (ST2+)cells were unchanged.In order to determine if the RA-responsive, CD8+ and IL-23R+ cells represented a single effector subset, we used sequential stripping/re-probing to deep phenotype the T cells. We identified a unique single CD8+ T-cell population that co-expressed RARα+, T-bet and IL23R, present in significantly increased numbers in gut biopsies from patients with GI-GvHD versus controls. This is consistent with a RA-responsive, IL-23-dependent, inflammatory Tc1 effector cell mediating tissue damage in human gut GvHD. Importantly, there was no increase in this T cell subset in skin biopsies of skin GvHD patients demonstrating tissue-specificity.Finally we determined the impact of exogenous RA exposure on T cell alloresponses in MLRs. RA significantly increased the proportion of alloproliferative effector CD8+ T cells co-expressing Tbet and gut-homing molecules, confirming that RA can directly potentiate human alloreactive CD8 T cell responses with capacity to home to the GI tract.Conclusions: This is the first data to demonstrate a role for RA at a tissue level in human GI-GvHD. Furthermore we have identified a gut specific CD8+ T cell subset which co-expresses RARα, T-bet, and IL-23R localised in areas of GI-damage likely to represent the RA-responsive effector cell. Therapeutic blockade of IL-23R could target this cellular response to prevent or treat GI-GvHD. DisclosuresGribben:Acerta: Honoraria; Janssen: Honoraria; Kite: Honoraria; Celgene: Honoraria; TG Therapeutics: Honoraria; Karyopharm: Honoraria; Pharmacyclics: Honoraria; Genentech/Roche: Honoraria; Abbvie: Honoraria.

Unconventional secretion of FABP4 by endosomes and secretory lysosomes.

An appreciation of the functional properties of the cytoplasmic fatty acid binding protein 4 (FABP4) has advanced with the recent demonstration that an extracellular form secreted by adipocytes regulates a wide range of physiological functions. Little, however, is known about the mechanisms that mediate the unconventional secretion of FABP4. Here, we demonstrate that FABP4 secretion is mediated by a membrane-bounded compartment, independent of the conventional endoplasmic reticulum-Golgi secretory pathway. We show that FABP4 secretion is also independent of GRASP proteins, autophagy, and multivesicular bodies but involves enclosure within endosomes and secretory lysosomes. We highlight the physiological significance of this pathway with the demonstration that an increase in plasma levels of FABP4 is inhibited by chloroquine treatment of mice. These findings chart the pathway of FABP4 secretion and provide a potential therapeutic means to control metabolic disorders associated with its dysregulated secretion.

Activation-Induced Conformational Changes of Dopamine D3 Receptor Promote the Formation of the Internal Water Channel

The atomic-level dopamine activation mechanism for transmitting extracellular ligand binding events through transmembrane helices to the cytoplasmic G protein remains unclear. In the present study, the complete dopamine D3 receptor (D3R), with a homology-modeled N-terminus, was constructed to dock different ligands to simulate conformational alterations in the receptor’s active and inactive forms during microsecond-timescale molecular dynamic simulations. In agonist-bound systems, the D3R N-terminus formed a “lid-like” structure and lay flat on the binding site opening, whereas in antagonist and inverse agonist-bound systems, the N-terminus exposed the binding cavity. Receptor activation was characterized using the different molecular switch residue distances, and G protein-binding site volumes. A continuous water pathway was observed only in the dopamine-Gαi-bound system. In the inactive D3Rs, water entry was hindered by the hydrophobic layers. Finally, a complete activation mechanism of D3R was proposed. Upon agonist binding, the “lid-like” conformation of the N-terminus induces a series of molecular switches to increase the volume of the D3R cytoplasmic binding part for G protein association. Meanwhile, water enters the transmembrane region inducing molecular switches to assist in opening the hydrophobic layers to form a continuous water channel, which is crucial for maintaining a fully active conformation for signal transduction.

Ulipristal acetate regulates perilipin-2 gene expression through progesterone receptor isoform B in uterine leiomyoma cells

Progesterone and its receptor (PR) exert a tumorigenic effect on leiomyoma cells. There are two functionally distinct isoforms of PR: PRA and PRB. Ulipristal acetate (UPA), a selective PR modulator, has been clinically shown to reduce the size of leiomyomas, although the exact mechanisms are unknown. Using an unbiased ChIP-sequencing technique, we previously uncovered perilipin-2 (PLIN2) as a novel PR target gene in primary human uterine leiomyoma cells. PLIN2, an adipose differentiation-related protein, is a cytoplasmic lipid droplet binding protein that plays an important role in lipid metabolism. PLIN2 has been shown to mediate cell proliferation, apoptosis, and fibrogenic gene expression in various cell types such as pancreatic and hepatic stellate cells; however, little is known about the role of PLIN2 in the regulation of the growth of human uterine leiomyomas. The present study aimed to test whether UPA regulates PLIN2 expression and to determine the function of PLIN2 in leiomyoma cells. Basic science. Myometrial and leiomyoma tissues were collected from premenopausal women undergoing hysterectomies. We examined the impact of UPA treatment on PLIN2 gene expression and its function in leiomyoma cells by depleting PLIN2 and PR expression via small interfering RNA (siRNA). We also characterized the PLIN2 gene expression patterns between leiomyoma and matched myometrial tissues. We found that 75% of subjects’ leiomyoma tissue expressed lower mRNA levels of PLIN2 compared to the adjacent myometrial tissue. We also found that UPA treatment induced PLIN2 expression in a time and dose-dependent manner in primary leiomyoma cells. When we knocked down PR to determine whether ulipristal treatment induced PLIN2 expression through PR, we found that PR siRNA dramatically decreased the total PR level to 20%; however, it only mildly decreased PRB expression (80% of control level), suggesting that PR siRNA may disproportionately deplete endogenous PRA more efficiently than PRB. Intriguingly, UPA treatment consistently stimulated PLIN2 expression in the control (scrambled) siRNA group. PLIN2 expression was further increased in the PR siRNA group, suggesting that UPA upregulated PLIN2 expression predominantly through PRB and that PRA may function to inhibit PRB-mediated PLIN2 transcriptional activity. Furthermore, knockdown of PLIN2 with siRNA significantly increased protein levels of proliferating cell nuclear antigen (a marker for cell proliferation). We present evidence that UPA stimulates PLIN2 expression and PLIN2 represses cell proliferation in primary uterine leiomyoma. Our findings also suggest that PRA and PRB may play differential roles in leiomyoma growth. We speculate that PLIN2 plays an important role in mediating the therapeutic effects of ulipristal in uterine leiomyomas.

Abstract LB-021: Impaired formation of homotypic cell-in-cell structures in human tumor cells lacking alpha-catenin expression

Abstract Majority of internal lumens in our bodies are covered with a layer of epithelial cells, whose integrity is critical for the organs to function properly. The integrity of epithelial tissues depends on intact adherens junctions (AJs), which is a multiple-components complex comprising cadherins, the transmembrane adhesion receptors, and their cytoplasmic binding proteins such as p120-catenin and b-catenin etc. Functional AJs is coupled with actin filaments through linker molecules, of which a-catenin and EPLIN are best characterized. Actin polymerization and actomyosin contraction regulated by Rho GTPases and their effectors play important role in AJs maintenance and remodeling. Aberrations, structural or functional, in AJs were associated with a number of pathological conditions, such as infection, inflammation and tumors and the like. Recent studies indicated that AJs mediated the formation of cell-in-cell structures (CICs). CICs refer to the cellular structures formed between viable cells, in which one or more cells exist inside other ones. Early records on CICs could be dated back to last century, when pathologists identified this type of unusual structures in human tumor samples10. Recent progress showed that cell-in-cell structures are rather complex than initially described, and could be classified into homotypic or heterotypic CICs based on the cells involved. Heterotypic CICs are usually formed by penetration of lymphocytes into tumor cells through processes like emperitosis. Homotypic CICs are formed between cells from same type, for example, epithelial cells inside epithelial cells. Mechanisms like entosis and homotypic cell cannibalism (HoCC) are responsible for this type of CICs formation. Once formed, CICs usually result in death of the internalized cells, which lead to the conception that CICs formation is a process of cell death. Limited researches identified extensive involvement of CICs in several important biological processes including development, immune homeostasis and tumor development and evolution etc. Recently, we and others found that formation of homotypic CICs by entosis was dependent on intact AJs and polarized actomyosin contraction. Tumor cells lacking epithelial cadherins (E and P-cadherin) failed to form CICs, moreover, re-expression of E- or P-cadherin could efficiently induce CICs in these cells, suggesting that disrupting AJs is a mechanism whereby tumor cells escape entosis-mediated CICs formation. In this work, we found that tumor cells deficient of a-catenin, a key component of functional AJs, also displayed impaired CICs formation, which could be fixed by restored expression of a -catenin. Therefore, tumor cells could escape entotic CICs formation by targeting multiple AJs components including E-/P-cadherin and a-catenin, and CICs formation by entosis may constitute a novel mechanism underlying the tumor suppressive function imposed by a -catenin. Note: This abstract was not presented at the meeting. Citation Format: Manna Wang, Xiangkai Ning, Ang Chen, Hongyan Huang, Qiang Sun, Zhaolie Chen, Li Ma, Xiaoning Wang. Impaired formation of homotypic cell-in-cell structures in human tumor cells lacking alpha-catenin expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-021. doi:10.1158/1538-7445.AM2017-LB-021

Modelling the free energy profile of the mitochondrial ADP/ATP carrier

The mitochondrial ADP/ATP carrier catalyses the equimolar exchange of adenosine di- and tri-phosphates. It operates by an alternating access mechanism in which a single substrate-binding site is made available either to the mitochondrial matrix or the intermembrane space through conformational changes. These changes are prevented in the absence of substrate by a large energy barrier due to the need for sequential disruption and formation of a matrix and cytoplasmic salt bridge network that are located on either side of the central cavity. In analogy to enzyme catalysis, substrate lowers the energy barrier by binding tighter in the intermediate state. Here we provide an in-silico kinetic model that captures the free energy profile of these conformational changes and treats the carrier as a nanomachine moving stochastically from the matrix to cytoplasmic conformation under the influence of thermal energy. The model reproduces the dependency of experimentally determined kcat and KM values on the cytoplasmic network strength with good quantitative accuracy, implying that it captures the transport mechanism and can provide a framework to understand the structure-function relationships of this class of transporter. The results show that maximum transport occurs when the interaction energies of the cytoplasmic network, matrix network and substrate binding are approximately equal such that the energy barrier is minimized. Consequently, the model predicts that there will be other interactions in addition to those of the cytoplasmic network that stabilise the matrix conformation of the ADP/ATP carrier.

Protein Phosphatase 1α enhances renal aldosterone signaling via mineralocorticoid receptor stabilization

Stimulation of the mineralocorticoid receptor (MR) by aldosterone controls several physiological parameters including blood pressure, inflammation or metabolism. We previously showed that MR turnover constitutes a crucial regulatory step in the responses of renal epithelial cells to aldosterone. Here, we identified Protein Phosphatase 1 alpha (PP1α), as a novel cytoplasmic binding partner of MR that promotes the receptor activity. The RT-PCR expression mapping of PP1α reveals a high expression in the kidney, particularly in the distal part of the nephron. At the molecular level, we demonstrate that PP1α inhibits the ubiquitin ligase Mdm2 by dephosphorylation, preventing its interaction with MR. This results in the accumulation of the receptor due to reduction of its proteasomal degradation and consequently a greater aldosterone-induced Na+ uptake by renal cells. Thus, our findings describe an original mechanism involving a phosphatase in the regulation of aldosterone signaling and provide new and important insights into the molecular mechanism underlying the MR turnover.

Reprogramming of the retinoic acid pathway in decidualizing human endometrial stromal cells.

Upon breaching of the endometrial surface epithelium, the implanting embryo embeds in the decidualizing stroma. Retinoic acid (RA), a metabolite of vitamin A, is an important morphogen during embryonic and fetal development, although the role of the RA pathway in the surrounding decidual cells is not understood. Here we show that decidual transformation of human endometrial stromal cells (HESCs) results in profound reprogramming of the RA signaling and metabolism pathways. Differentiating HESCs downregulate the intracellular carrier proteins CRABP2 and FABP5, responsible for transfer and binding of RA to the nuclear receptors RAR and PPARβ/δ, respectively. Furthermore, the expression of RAR, the receptor that mediates the pro-apoptotic effects of RA, was also inhibited. By contrast, PPARβ/δ, which transduces the differentiation responses of RA, was upregulated. Decidualization was also associated with increased expression of retinol-binding protein 4 (RBP4) and various enzymes involved in the metabolism of RA and its precursor, retinaldehyde (Rald), including CYP26A1, DHRS3, and RDH12. Exposure of differentiating HESCs to RA or Rald reversed the inhibition of the CRABP2-RAR pathway, perturbed the expression of decidual marker genes and triggered cell death. Taken together, the data demonstrate that decidualizing HESCs silence RA signaling by downregulating key cytoplasmic binding proteins and by increasing retinoid metabolism. However, excessive RA exposure is toxic for decidual cells and triggers a response that may lead to pregnancy failure.

Implications for the Light-Driven Chloride Ion Transport Mechanism of Nonlabens marinus Rhodopsin 3 by Its Photochemical Characteristics.

Several new retinal-based photoreceptor proteins that act as light-driven electrogenic halide ion pumps have recently been discovered. Some of them, called "NTQ" rhodopsins, contain a conserved Asn-Thr-Gln motif in the third or C-helix. In this study, we investigated the photochemical characteristics of an NTQ rhodopsin, Nonlabens marinus rhodopsin 3 (NM-R3), which was discovered in the N. marinus S1-08T strain, using static and time-resolved spectroscopic techniques. We demonstrate that NM-R3 binds a Cl- in the vicinity of the retinal chromophore accompanied by a spectral blueshift from 568 nm in the absence of Cl- to 534 nm in the presence of Cl-. From the Cl- concentration dependence, we estimated the affinity (dissociation constant, Kd) for Cl- in the original state as 24 mM, which is ca. 10 times weaker than that of archaeal halorhodopsins but ca. 3 times stronger than that of a marine bacterial Cl- pumping rhodopsin (C1R). NM-R3 showed no dark-light adaptation of the retinal chromophore and predominantly possessed an all-trans-retinal, which is responsible for the light-driven Cl- pump function. Flash-photolysis experiments suggest that NM-R3 passes through five or six photochemically distinct intermediates (K, L(N), O1, O2, and NM-R3'). From these results, we assume that the Cl- is released and taken up during the L(N)-O1 transition from a transiently formed cytoplasmic (CP) binding site and the O2-NM-R3' or the NM-R3'-original NM-R3 transitions from the extracellular (EC) side, respectively. We propose a mechanism for the Cl- transport by NM-R3 based on our results and its recently reported crystal structure.

Siderophore transport by MmpL5-MmpS5 protein complex in Mycobacterium tuberculosis

Iron is an essential metal ion required for the various physiological activities of bacteria. The pathogenic bacteria remain dependent on the host cell for their iron requirements and evolved with specialized scavenging machinery in the form of iron chelating siderophores. Mycobacterium tuberculosis has two types of siderophore molecules, mycobactin and carboxymycobactin. These are synthesized inside bacterial cells and need to be transported outside by specialized membrane associated proteins. MmpL5-MmpS5 (mycobacterial membrane protein large5-mycobacterial membrane protein small5) complex has been linked to the export of non-ferrated siderophores to extracellular environment but the precise molecular mechanism involved was largely unknown. We have investigated the association of MmpL5 with mycobactin synthesis and transport associated proteins using system wide protein-protein interaction network. Insights of mycobactin transport mechanism by MmpL5-MmpS5 complex was explored using docking and molecular dynamics simulations. The MmpL5 has association with many proteins with reported roles in iron acquisition or mycobactin biosynthesis. The molecular dynamics simulation analysis after mycobactin docking into MmpL5 binding pockets showed that at cytoplasmic binding site, mycobactin could move towards the central channel of efflux pump and at periplasmic binding site towards the periplasm. MmpL5 was observed to carry out uptake of mycobactin from the cytoplasm and its release into the periplasmic space and MmpS5 was found to facilitate the periplasmic release of mycobactin and enhancement in the transport function of MmpL5. The mycobactin export is an attractive target for drug discovery and it may be carried out by inhibiting the MmpL5 protein's transport function.

Lineage-specific roles of the cytoplasmic polyadenylation factor CPEB4 in the regulation of melanoma drivers.

Nuclear 3'-end-polyadenylation is essential for the transport, stability and translation of virtually all eukaryotic mRNAs. Poly(A) tail extension can also occur in the cytoplasm, but the transcripts involved are incompletely understood, particularly in cancer. Here we identify a lineage-specific requirement of the cytoplasmic polyadenylation binding protein 4 (CPEB4) in malignant melanoma. CPEB4 is upregulated early in melanoma progression, as defined by computational and histological analyses. Melanoma cells are distinct from other tumour cell types in their dependency on CPEB4, not only to prevent mitotic aberrations, but to progress through G1/S cell cycle checkpoints. RNA immunoprecipitation, sequencing of bound transcripts and poly(A) length tests link the melanoma-specific functions of CPEB4 to signalling hubs specifically enriched in this disease. Essential in these CPEB4-controlled networks are the melanoma drivers MITF and RAB7A, a feature validated in clinical biopsies. These results provide new mechanistic links between cytoplasmic polyadenylation and lineage specification in melanoma.

The Structure of the Periplasmic Sensor Domain of the Histidine Kinase CusS Shows Unusual Metal Ion Coordination at the Dimeric Interface.

In bacteria, two-component systems act as signaling systems to respond to environmental stimuli. Two-component systems generally consist of a sensor histidine kinase and a response regulator, which work together through histidyl-aspartyl phosphorelay to result in gene regulation. One of the two-component systems in Escherichia coli, CusS-CusR, is known to induce expression of cusCFBA genes at increased periplasmic Cu(I) and Ag(I) concentrations to help maintain metal ion homeostasis. CusS is a membrane-associated histidine kinase with a periplasmic sensor domain connected to the cytoplasmic ATP binding and catalytic domains through two transmembrane helices. The mechanism of how CusS senses increasing metal ion concentrations and activates CusR is not yet known. Here, we present the crystal structure of the Ag(I)-bound periplasmic sensor domain of CusS at a resolution of 2.15 Å. The structure reveals that CusS forms a homodimer with four Ag(I) binding sites per dimeric complex. Two symmetric metal binding sites are found at the dimeric interface, which are each formed by two histidines and one phenylalanine with an unusual cation-π interaction. The other metal ion binding sites are in a nonconserved region within each monomer. Functional analyses of CusS variants with mutations in the metal sites suggest that the metal ion binding site at the dimer interface is more important for function. The structural and functional data provide support for a model in which metal-induced dimerization results in increases in kinase activity in the cytoplasmic domains of CusS.

Total cellular protein presence of the transcription factor IRF8 does not necessarily correlate with its nuclear presence

The transcription factor interferon regulatory factor-8 (IRF8) plays an essential role in myeloid differentiation and lineage commitment, based largely on molecular and genetic studies. The detection of IRF8 in specific cell populations by flow cytometry (FCM) has the potential to provide new insights into normal and pathologic myelopoiesis, but critical validation of this protein-based approach, particularly in human samples, is lacking. In this study, the assessment of total cellular IRF8 presence was compared to its specific nuclear presence as assessed by imaging flow cytometry (IFC) analysis. Peptide neutralization of the IRF8-specific antibody that has been predominantly used to date in the literature served as a negative control for the immunofluorescent labeling. Expression of total IRF8 was analyzed by total cellular fluorescence analogous to the mean fluorescence intensity readout of conventional FCM. Additionally, specific nuclear fluorescence and the similarity score between the nuclear image (DAPI) and the corresponding IRF8 image for each cell were analyzed as parameters for nuclear localization of IRF8. IFC showed that peptide blocking eliminated binding of the IRF8 antibody in the nucleus. It also reduced cytoplasmic binding of the antibody but not to the extent observed in the nucleus. In agreement with the similarity score data, the total cellular IRF8 as well as nuclear IRF8 intensities decreased with peptide blocking. In healthy donor peripheral blood subpopulations and a positive control cell line (THP-1), the assessment of IRF8 by total cellular presence correlated well with its specific nuclear presence and correlated with the known distribution of IRF8 in these cells. In clinical samples of myeloid-derived suppressors cells derived from patients with renal carcinoma, however, total cellular IRF8 did not necessarily correlate with its nuclear presence. Discordance was primarily associated with peptide blocking having a proportionally greater effect on the IRF8 nuclear localization versus total fluorescence assessment. The data thus indicate that IRF8 can have cytoplasmic presence and that during disease its nuclear-cytoplasmic distribution may be altered, which may provide a basis for potential myeloid defects during certain pathologies.

Epithelial-mesenchymal transition and nuclear β-catenin induced by conditional intestinal disruption of Cdh1 with Apc is E-cadherin EC1 domain dependent.

Two important protein-protein interactions establish E-cadherin (Cdh1) in the adhesion complex; homophilic binding via the extra-cellular (EC1) domain and cytoplasmic tail binding to β-catenin. Here, we evaluate whether E-cadherin binding can inhibit β-catenin when there is loss of Adenomatous polyposis coli (APC) from the β-catenin destruction complex. Combined conditional loss of Cdh1 and Apc were generated in the intestine, intestinal adenoma and adenoma organoids. Combined intestinal disruption (Cdh1fl/flApcfl/flVil-CreERT2) resulted in lethality, breakdown of the intestinal barrier, increased Wnt target gene expression and increased nuclear β-catenin localization, suggesting that E-cadherin inhibits β-catenin. Combination with an intestinal stem cell Cre (Lgr5CreERT2) resulted in ApcΔ/Δ recombination and adenoma, but intact Cdh1fl/fl alleles. Cultured ApcΔ/ΔCdh1fl/fl adenoma cells infected with adenovirus-Cre induced Cdh1fl/fl recombination (Cdh1Δ/Δ), disruption of organoid morphology, nuclear β-catenin localization, and cells with an epithelial-mesenchymal phenotype. Complementation with adenovirus expressing wild-type Cdh1 (Cdh1-WT) rescued adhesion and β-catenin membrane localization, yet an EC1 specific double mutant defective in homophilic adhesion (Cdh1-MutW2A, S78W) did not. These data suggest that E-cadherin inhibits β-catenin in the context of disruption of the APC-destruction complex, and that this function is also EC1 domain dependent. Both binding functions of E-cadherin may be required for its tumour suppressor activity.

Reciprocal Control of Thyroid Binding and the Pipecolate Pathway in the Brain.

Thyroid hormones have long been known to play an essential role in brain growth and development, with cytoplasmic thyroid hormone binding proteins (THBPs) playing a critical role in thyroid hormone bioavailability. A major mammalian THBP is μ-crystallin (CRYM), which was originally characterized by its ability to strongly bind thyroid hormones in an NADPH-dependent fashion. However, in 2011 it was discovered that CRYM is also an enzyme, namely ketimine reductase (KR), which catalyzes the NAD(P)H-dependent reduction of -C=N- (imine) double bonds of a number of cyclic ketimine substrates including sulfur-containing cyclic ketimines. The enzyme activity was also shown to be potently inhibited by thyroid hormones, thus suggesting a novel reciprocal relationship between enzyme catalysis and thyroid hormone bioavailability. KR is involved in a number of amino acid metabolic pathways. However, the best documented biological function of KR is its role as a ∆1-piperideine-2-carboxylate (P2C) reductase in the pipecolate pathway of lysine metabolism. The pipecolate pathway is the main L-lysine degradation pathway in the adult brain, whereas the saccharopine pathway predominates in extracerebral tissues and in infant brain, suggesting that KR has evolved to perform specific and important roles in neural development and function. The potent regulation of KR activity by thyroid hormones adds further weight to this suggestion. KR is also involved in L-ornithine/L-glutamate/L-proline metabolism as well as sulfur-containing amino acid metabolism. This review describes the pipecolate pathway and recent discoveries related to mammalian KR function, which have important implications in normal and pathological brain functions.