Journal of Biological Chemistry

doi: 10.1016/s0021-9258(20)43256-9pmid: N/A

doi: 10.1016/s0021-9258(20)43255-7pmid: N/A

Sutherland, Cindy;Walsh, Michael P.

doi: 10.1074/jbc.m112.371609pmid: 22661704

<p>The principal signal to activate smooth muscle contraction is phosphorylation of the regulatory light chains of myosin (LC₂₀) at Ser¹⁹ by Ca²⁺/calmodulin-dependent myosin light chain kinase. Inhibition of myosin light chain phosphatase leads to Ca²⁺-independent phosphorylation at both Ser¹⁹ and Thr¹⁸ by integrin-linked kinase and/or zipper-interacting protein kinase. The functional effects of phosphorylation at Thr¹⁸ on steady-state isometric force and relaxation rate were investigated in Triton-skinned rat caudal arterial smooth muscle strips. Sequential phosphorylation at Ser¹⁹ and Thr¹⁸ was achieved by treatment with adenosine 5′-<i>O</i>-(3-thiotriphosphate) in the presence of Ca²⁺, which induced stoichiometric thiophosphorylation at Ser¹⁹, followed by microcystin (phosphatase inhibitor) in the absence of Ca²⁺, which induced phosphorylation at Thr¹⁸. Phosphorylation at Thr¹⁸ had no effect on steady-state force induced by Ser¹⁹ thiophosphorylation. However, phosphorylation of Ser¹⁹ or both Ser¹⁹ and Thr¹⁸ to comparable stoichiometries (0.5 mol of P_i/mol of LC₂₀) and similar levels of isometric force revealed differences in the rates of dephosphorylation and relaxation following removal of the stimulus: <i>t</i>_½ values for dephosphorylation were 83.3 and 560 s, and for relaxation were 560 and 1293 s, for monophosphorylated (Ser¹⁹) and diphosphorylated LC₂₀, respectively. We conclude that phosphorylation at Thr¹⁸ decreases the rates of LC₂₀ dephosphorylation and smooth muscle relaxation compared with LC₂₀ phosphorylated exclusively at Ser¹⁹. These effects of LC₂₀ diphosphorylation, combined with increased Ser¹⁹ phosphorylation (Ca²⁺-independent), may underlie the hypercontractility that is observed in response to certain physiological contractile stimuli, and under pathological conditions such as cerebral and coronary arterial vasospasm, intimal hyperplasia, and hypertension.</p><p><b>Background:</b> The regulatory light chains of smooth muscle myosin are phosphorylated at Ser¹⁹ and Thr¹⁸.</p><p><b>Results:</b> Phosphorylation at Thr¹⁸ does not increase force elicited by Ser¹⁹ phosphorylation, but reduces the rate of relaxation.</p><p><b>Conclusion:</b> Diphosphorylation slows relaxation compared with monophosphorylation at Ser¹⁹.</p><p><b>Significance:</b> Knowledge of the functional effects of myosin diphosphorylation is important for understanding the underlying causes of hypercontractility.</p>

Karlberg, Tobias;Thorsell, Ann-Gerd;Kallas, Åsa;Schüler, Herwig

doi: 10.1074/jbc.m112.379289pmid: 22661712

<p>ADP-ribosylation is involved in the regulation of DNA repair, transcription, and other processes. The 18 human ADP-ribose transferases with diphtheria toxin homology include ARTD1/PARP1, a cancer drug target. Knowledge of other family members may guide therapeutics development and help evaluate potential drug side effects. Here, we present the crystal structure of human ARTD15/PARP16, a previously uncharacterized enzyme. ARTD15 features an α-helical domain that packs against its transferase domain without making direct contact with the NAD⁺-binding crevice or the donor loop. Thus, this novel domain does not resemble the regulatory domain of ARTD1. ARTD15 displays auto-mono(ADP-ribosylation) activity and is affected by canonical poly(ADP-ribose) polymerase inhibitors. These results add to a framework that will facilitate research on a medically important family of enzymes.</p><p><b>Background:</b> ADP-ribose transferases ARTD1–3/PARP1–3 have an α-helical domain that closes over the NAD⁺-binding site.</p><p><b>Results:</b> Human ARTD15/PARP16 is a mono(ADP-ribose) transferase with a novel α-helical domain that interacts with a catalytic domain loop.</p><p><b>Conclusion:</b> The ARTD15 transferase domain is likely regulated by effector binding to the adjacent helical domain.</p><p><b>Significance:</b> This provides a basis for understanding the enzymatic mechanism of this previously uncharacterized enzyme.</p>

Chen, Youjun;Chalouni, Cecile;Tan, Christine;Clark, Robyn;Venook, Rayna;Ohri, Rachana;Raab, Helga;Firestein, Ron;Mallet, William;Polakis, Paul

doi: 10.1074/jbc.m112.361485pmid: 22613716

<p>Melanocytes uniquely express specialized genes required for pigment formation, some of which are maintained following their transformation to melanoma. Here we exploit this property to selectively target melanoma with an antibody drug conjugate (ADC) specific to PMEL17, the product of the SILV pigment-forming gene. We describe new PMEL17 antibodies that detect the endogenous protein. These antibodies help define the secretory fate of PMEL17 and demonstrate its utility as an ADC target. Although newly synthesized PMEL17 is ultimately routed to the melanosome, we find substantial amounts accessible to our antibodies at the cell surface that undergo internalization and routing to a LAMP1-enriched, lysosome-related organelle. Accordingly, an ADC reactive with PMEL17 exhibits target-dependent tumor cell killing <i>in vitro</i> and <i>in vivo</i>.</p><p><b>Background:</b> A search for cell surface proteins amenable to antibody drug conjugate (ADC) therapy was performed.</p><p><b>Results:</b> Expression of PMEL17 was highly restricted to melanoma cells, and an ADC directed against it was efficacious.</p><p><b>Conclusion:</b> PMEL17 is an attractive target for ADC therapy in melanoma.</p><p><b>Significance:</b> Intracellular transmembrane proteins that transit the cell surface represent a new class of targets for ADCs.</p>

Chucair-Elliott, Ana J.;Elliott, Michael H.;Wang, Jiangang;Moiseyev, Gennadiy P.;Ma, Jian-Xing;Politi, Luis E.;Rotstein, Nora P.;Akira, Shizuo;Uematsu, Satoshi;Ash, John D.

doi: 10.1074/jbc.m112.378240pmid: 22645143

<p>Leukemia inhibitory factor (LIF), an interleukin-6 family neurocytokine, is up-regulated in response to different types of retinal stress and has neuroprotective activity through activation of the gp130 receptor/STAT3 pathway. We observed that LIF induces rapid, robust, and sustained activation of STAT3 in both the retina and retinal pigmented epithelium (RPE). Here, we tested whether LIF-induced STAT3 activation within the RPE can down-regulate RPE65, the central enzyme in the visual cycle that provides the 11-<i>cis</i>-retinal chromophore to photoreceptors <i>in vivo</i>. We generated conditional knock-out mice to specifically delete STAT3 or gp130 in RPE, retina, or both RPE and retina. After intravitreal injection of LIF, we analyzed the expression levels of visual cycle genes and proteins, isomerase activity of RPE65, levels of rhodopsin protein, and the rates of dark adaptation and rhodopsin regeneration. We found that RPE65 protein levels and isomerase activity were reduced and recovery of bleachable rhodopsin was delayed in LIF-injected eyes. In mice with functional gp130/STAT3 signaling in the retina, rhodopsin protein was also reduced by LIF. However, the LIF-induced down-regulation of RPE65 required a functional gp130/STAT3 cascade intrinsic to RPE. Our data demonstrate that a single cytokine, LIF, can simultaneously and independently affect both RPE and photoreceptors through the same signaling cascade to reduce the generation and utilization of 11-<i>cis</i>-retinal.</p><p><b>Background:</b> Neurocytokines (LIF and CNTF) mediate photoreceptor protection and down-regulation of phototransduction.</p><p><b>Results:</b> LIF down-regulates the visual cycle decreasing RPE65 expression and activity through activation of STAT3 in RPE.</p><p><b>Conclusion:</b> The gp130/STAT3 pathway is independently modulated in RPE and retina for coordinated control of visual cycle activity.</p><p><b>Significance:</b> A single, endogenous paracrine factor (LIF) can stimulate RPE cells to reduce production of 11-<i>cis</i>-retinal.</p>

Jeanes, Alexa I.;Wang, Pengbo;Moreno-Layseca, Paulina;Paul, Nikki;Cheung, Julia;Tsang, Ricky;Akhtar, Nasreen;Foster, Fiona M.;Brennan, Keith;Streuli, Charles H.

doi: 10.1074/jbc.m112.360834pmid: 22511753

<p>Understanding how cell cycle is regulated in normal mammary epithelia is essential for deciphering defects of breast cancer and therefore for developing new therapies. Signals provided by both the extracellular matrix and growth factors are essential for epithelial cell proliferation. However, the mechanisms by which adhesion controls cell cycle in normal epithelia are poorly established. In this study, we describe the consequences of removing the β1-integrin gene from primary cultures of mammary epithelial cells <i>in situ</i>, using CreER. Upon β1-integrin gene deletion, the cells were unable to progress efficiently through S-phase, but were still able to undergo collective two-dimensional migration. These responses are explained by the presence of β3-integrin in β1-integrin-null cells, indicating that integrins containing different β-subunits exert differential control on mammary epithelial proliferation and migration. β1-Integrin deletion did not inhibit growth factor signaling to Erk or prevent the recruitment of core adhesome components to focal adhesions. Instead the S-phase arrest resulted from defective Rac activation and Erk translocation to the nucleus. Rac inhibition prevented Erk translocation and blocked proliferation. Activated Rac1 rescued the proliferation defect in β1-integrin-depleted cells, indicating that this GTPase is essential in propagating proliferative β1-integrin signals. These results show that β1-integrins promote cell cycle in mammary epithelial cells, whereas β3-integrins are involved in migration.</p><p><b>Background:</b> Integrin-mediated ECM adhesion is required for mammary epithelial proliferation, but the mechanism is not known.</p><p><b>Results:</b> Gene deletion studies show that β1-integrin-null mammary epithelial cells retain β3-integrins and the ability to undergo two-dimensional migration, and Rac1 rescues their proliferation defect.</p><p><b>Conclusion:</b> β1-Integrins uniquely control proliferation in mammary cells via Rac1, whereas β3-integrins support two-dimensional migration.</p><p><b>Significance:</b> Specific β-integrin-containing adhesions determine different cell-fate responses.</p>

Sato, Sho;Kawamoto, Jun;Sato, Satoshi B.;Watanabe, Bunta;Hiratake, Jun;Esaki, Nobuyoshi;Kurihara, Tatsuo

doi: 10.1074/jbc.m111.318311pmid: 22648406

<p>In this study, we found that phospholipids containing an eicosapentaenyl group form a novel membrane microdomain at the cell division site of a Gram-negative bacterium, <i>Shewanella livingstonensis</i> Ac10, using chemically synthesized fluorescent probes. The occurrence of membrane microdomains in eukaryotes and prokaryotes has been demonstrated with various imaging tools for phospholipids with different polar headgroups. However, few studies have focused on the hydrocarbon chain-dependent localization of membrane-resident phospholipids <i>in vivo</i>. We previously found that lack of eicosapentaenoic acid (EPA), a polyunsaturated fatty acid found at the <i>sn</i>-2 position of glycerophospholipids, causes a defect in cell division after DNA replication of <i>S. livingstonensis</i> Ac10. Here, we synthesized phospholipid probes labeled with a fluorescent 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) group to study the localization of EPA-containing phospholipids by fluorescence microscopy. A fluorescent probe in which EPA was bound to the glycerol backbone via an ester bond was found to be unsuitable for imaging because EPA was released from the probe by <i>in vivo</i> hydrolysis. To overcome this problem, we synthesized hydrolysis-resistant ether-type phospholipid probes. Using these probes, we found that the fluorescence localized between two nucleoids at the cell center during cell division when the cells were grown in the presence of the eicosapentaenyl group-containing probe (<i>N</i>-NBD-1-oleoyl-2-eicosapentaenyl-<i>sn</i>-glycero-3-phosphoethanolamine), whereas this localization was not observed with the oleyl group-containing control probe (<i>N</i>-NBD-1-oleoyl-2-oleyl-<i>sn</i>-glycero-3-phosphoethanolamine). Thus, phospholipids containing an eicosapentaenyl group are specifically enriched at the cell division site. Formation of a membrane microdomain enriched in EPA-containing phospholipids at the nucleoid occlusion site probably facilitates cell division.</p><p><b>Background:</b> Phospholipids containing polyunsaturated hydrocarbon chains play important roles in various biological membranes.</p><p><b>Results:</b> Fluorescence microscopic analysis with a chemically synthesized fluorescent probe showed localization of phospholipids containing an eicosapentaenyl group at the bacterial cell division site.</p><p><b>Conclusion:</b> Phospholipids containing polyunsaturated hydrocarbon chains form a membrane microdomain at the cell center, probably to promote cell division.</p><p><b>Significance:</b> Hydrocarbon chain-dependent microdomain formation was visualized <i>in vivo</i>.</p>

Meech, Robyn;Rogers, Anne;Zhuang, Lizhe;Lewis, Benjamin C.;Miners, John O.;Mackenzie, Peter I.

doi: 10.1074/jbc.m112.343608pmid: 22621930

<p>Recent studies in this laboratory characterized the UGT3A family enzymes, UGT3A1 and UGT3A2, and showed that neither uses the traditional UDP-glycosyltransferase UGT co-substrate UDP-glucuronic acid. Rather, UGT3A1 uses GlcNAc as preferred sugar donor and UGT3A2 uses UDP-Glc. The enzymatic characterization of UGT3A mutants, structural modeling, and multispecies gene analysis have now been employed to identify a residue within the active site of these enzymes that confers their unique sugar preferences. An asparagine (Asn-391) in the UGT signature sequence of UGT3A1 is necessary for utilization of UDP-GlcNAc. Conversely, a phenylalanine (Phe-391) in UGT3A2 favors UDP-Glc use. Mutation of Asn-391 to Phe in UGT3A1 enhances its ability to utilize UDP-Glc and completely inhibits its ability to use UDP-GlcNAc. An analysis of homology models docked with UDP-sugar donors indicates that Asn-391 in UGT3A1 is able to accommodate the <i>N</i>-acetyl group on C2 of UDP-GlcNAc so that the anomeric carbon atom (C1) is optimally situated for catalysis involving His-35. Replacement of Asn with Phe at position 391 disrupts this catalytically productive orientation of UDP-GlcNAc but allows a more optimal alignment of UDP-Glc for sugar donation. Multispecies sequence analysis reveals that only primates possess UGT3A sequences containing Asn-391, suggesting that other mammals may not have the capacity to <i>N</i>-acetylglucosaminidate small molecules. In support of this hypothesis, Asn-391-containing UGT3A forms from two non-human primates were found to use UDP-GlcNAc, whereas UGT3A isoforms from non-primates could not use this sugar donor. This work gives new insight into the residues that confer sugar specificity to UGT family members and suggests a primate-specific innovation in glycosidation of small molecules.</p><p><b>Background:</b> Conjugation of sugars to chemicals by (UDP-glycosyltransferases) UGTs is a critical detoxification mechanism.</p><p><b>Results:</b> A single amino acid defines the differential sugar specificities of two related UGTs.</p><p><b>Conclusion:</b> The change of a single amino acid during primate evolution has generated a new capacity for small molecule glycosidation.</p><p><b>Significance:</b> Determinants of UGT sugar selectivity are currently poorly understood; novel glycosidation pathways may have important metabolic roles.</p>

Montserrat, Nuria;Ramírez-Bajo, María José;Xia, Yun;Sancho-Martinez, Ignacio;Moya-Rull, Daniel;Miquel-Serra, Laia;Yang, Shenglian;Nivet, Emmanuel;Cortina, Carme;González, Federico;Belmonte, Juan Carlos Izpisua;Campistol, Josep M.

doi: 10.1074/jbc.m112.350413pmid: 22613719

<p>The tubular epithelium of the kidney is susceptible to injury from a number of different causes, including inflammatory and immune disorders, oxidative stress, and nephrotoxins, among others. Primary renal epithelial cells remain one of the few tools for studying the biochemical and physiological characteristics of the renal tubular system. Nevertheless, differentiated primary cells are not suitable for recapitulation of disease properties that might arise during embryonic kidney formation and further maturation. Thus, cellular systems resembling kidney characteristics are in urgent need to model disease as well as to establish reliable drug-testing platforms. Induced pluripotent stem cells (iPSCs) bear the capacity to differentiate into every cell lineage comprising the adult organism. Thus, iPSCs bring the possibility for recapitulating embryonic development by directed differentiation into specific lineages. iPSC differentiation ultimately allows for both disease modeling <i>in vitro</i> and the production of cellular products with potential for regenerative medicine. Here, we describe the rapid, reproducible, and highly efficient generation of iPSCs derived from endogenous kidney tubular renal epithelial cells with only two transcriptional factors, <i>OCT4</i> and <i>SOX2</i>. Kidney-derived iPSCs may provide a reliable cellular platform for the development of kidney differentiation protocols allowing drug discovery studies and the study of kidney pathology.</p><p><b>Background:</b> The generation of human-induced pluripotent stem cells (iPS) has raised expectations for disease modeling, drug discovery, and cell therapy.</p><p><b>Results:</b> VP16-polycistronic vectors display enhanced reprogramming capacity.</p><p><b>Conclusion:</b> Primary tubular renal cells are amenable for iPSC reprogramming in the absence of oncogenes.</p><p><b>Significance:</b> Kidney-derived iPSCs provide a reliable cellular platform for the study of kidney pathology and drug discovery studies.</p>