A cleavable affinity biotinylating agent reveals a retinoid binding role for RPE65

Retinal pigment epithelial (RPE) membranes contain the full biochemical apparatus capable of processing all-trans-retinol (vitamin A) into 11-cis-retinal, the visual chromophore. As many of these proteins are integral membrane proteins and resistant to traditional methods of identification, alternate methods of identifying these proteins are sought. The approach described here involves affinity biotinylation with alkali cleavable linkers. A vitamin A containing affinity-labeling haloacetate is described which facilitates the identification of retinoid binding proteins (RBPs). Treatment of crude bovine RPE membranes with (3R)-3-[boc-lys(biotinyl)-O]-all-trans-retinol chloroacetate 1 in the low micromolar range led to the specific labeling of RPE65 and lecithin retinol acyltransferase (LRAT). Only RPE65 is labeled at 5 microM 1 at 4 degrees C. Labeled RPE65 was readily isolated by binding the labeled protein to avidin-containing beads, followed by cleavage of the protein from the beads at pH 11. Trypsin digestion of RPE65 modified by 1, followed by mass spectrometry, demonstrates that C231 and C448 are alkylated by 1. These studies validate the approach that was used, and furthermore demonstrate that RPE65, a major membrane-associated protein of the RPE, is a RBP.     

Lecithin retinol acyltransferase forms functional homodimers

Membrane-bound lecithin retinol acyltransferase (LRAT), an essential enzyme in vitamin A processing, catalyzes the formation of retinyl esters from vitamin A and lecithin. Cloned and expressed LRAT has a molecular mass of 25.3 kDa. The enzyme is not homologous to known enzymes and is, therefore, of substantial interest mechanistically. Along these lines, the functional protomeric state of LRAT is of importance. Gel electrophoretic studies on LRAT in the presence of SDS and disulfide reducing agents show the expected 25 kDa monomer. However, gel electrophoresis in the absence of a reducing agent and/or strong denaturing conditions reveals substantial dimer formation. LRAT monomers can be efficiently and irreversibly cross-linked by thiol reactive bismaleimides in retinal pigment epithelial (RPE) membranes generating LRAT homodimers. Cross-linked LRAT homodimers are fully active catalytically. The experiments suggest that LRAT monomers interact in membranes and form functional homodimers through protein-protein interactions and disulfide bond formation.     

Zinc mediates assembly of the T1 domain of the voltage-gated K channel 4.2

An intermolecular Zn(2+)-binding site was identified in the structure of the T1 domain of the Shaw-type potassium channels (aKv3.1). T1 is a BTB/POZ-type domain responsible for the ordered assembly of voltage-gated potassium channels and interactions with other macromolecules. In this structure, a Zn(2+) ion was found to be coordinated between each of the four assembly interfaces of the T1 tetramer by three Cys and one His encoded in the sequence motif (HX(5)CX(20)CC) of the T1 domain. This sequence motif is conserved among all non-Shaker-type voltage-dependent potassium (Kv) channels, but not in Shaker-type channels. The presence of this conserved Zn(2+)-binding site is a primary molecular determinant that distinguishes the tetrameric assembly of non-Shaker Kv channel subunits from that of Shaker channels. We report here that tetramerization of the Shal (rKv4.2) T1 in solution requires the presence of Zn(2+), and the addition/removal of Zn(2+) reversibly switches the protein between a stable tetrameric or monomeric state. We further show that the conversion from tetramers to monomers is profoundly pH-dependent: as the solution pH gets lower, the dissociation rate increases significantly. The unfolding energy of the T1 tetramer as a measure of the conformational stability of the structure is also pH-dependent. Surprisingly, at a lower pH we observe a distinctly altered conformational state of the T1 tetramer trapped during the process of unfolding of the T1 tetramer in the presence of Zn(2+). The conformational alteration may be responsible for increased rate of dissociation at lower pH by allowing Zn(2+) to be removed more effectively by EDTA. The ability of the T1 domain to adopt stable alternative conformations may be essential to its function as a protein-protein interaction/signaling domain to modulate the ion conduction properties of intact full-length Kv channels.     

The human cytomegalovirus UL44 protein is a substrate for the UL97 protein kinase

The human cytomegalovirus UL97 protein is an unusual protein kinase that is able to autophosphorylate and to phosphorylate certain exogenous substrates, including nucleoside analogs such as ganciclovir. However, no natural substrate of UL97 in infected cells has been identified. We report here that recombinant UL44 protein became radiolabeled when incubated with recombinant UL97 and [(32)P]ATP and that both proteins could be coimmunoprecipitated by an antibody that recognizes either protein. Subsequent studies showed that highly purified, recombinant UL97 phosphorylated purified, recombinant UL44. This phosphorylation occurred on serine and threonine residues and was sensitive to inhibition by maribavir and to a mutation that inactivates UL97 catalytic activity. Two-dimensional gel electrophoresis revealed the absence of specific phosphorylated forms of UL44 in immunoprecipitates from lysates of cells infected with a UL97 null mutant virus or with wild-type virus in the presence of maribavir. The results indicate that UL97 is sufficient to phosphorylate UL44 in vitro and is necessary for the normal phosphorylation of UL44 in infected cells. This strongly suggests that UL44 is a natural substrate of UL97.     

The GanB Galpha-protein negatively regulates asexual sporulation and plays a positive role in conidial germination in Aspergillus nidulans

We isolated the ganB gene encoding the Galpha-protein homolog from Aspergillus nidulans. To investigate the cellular function of GanB, various mutant strains were isolated. Deletion of constitutively inactive ganB mutants showed conidiation and derepressed brlA expression in a submerged culture. Constitutive activation of GanB caused a reduction in hyphal growth and a severe defect in asexual sporulation. We therefore propose that GanB may negatively regulate asexual sporulation through the BrlA pathway. In addition, deletion or constitutive inactivation of GanB reduced germination rate while constitutive activation led to precocious germination. Furthermore, conidia of a constitutively active mutant could germinate even without carbon source. Taken together, these results indicated that GanB plays a positive role during germination, possibly through carbon source sensing, and negatively regulates asexual conidiation in A. nidulans.     

A palmitoylation switch mechanism in the regulation of the visual cycle

RPE65 is essential for the biosynthesis of 11-cis-retinal, the chromophore of rhodopsin. Here, we show that the membrane-associated form (mRPE65) is triply palmitoylated and is a chaperone for all-trans-retinyl esters, allowing their entry into the visual cycle for processing into 11-cis-retinal. The soluble form of RPE65 (sRPE65) is not palmitoylated and is a chaperone for vitamin A, rather than all-trans-retinyl esters. Thus, the palmitoylation of RPE65 controls its ligand binding selectivity. The two chaperones are interconverted by lecithin retinol acyl transferase (LRAT) acting as a molecular switch. Here mRPE65 is a palmitoyl donor, revealing a new acyl carrier protein role for palmitoylated proteins. When chromophore synthesis is not required, mRPE65 is converted into sRPE65 by LRAT, and further chromophore synthesis is blocked. The studies reveal new roles for palmitoylated proteins as molecular switches and LRAT as a palmitoyl transferase whose role is to catalyze the mRPE65 to sRPE65 conversion.     

Characterisation of<Emphasis Type="Italic">fusarium graminearum</Emphasis> and<Emphasis Type="Italic">F. culmorum</Emphasis> isolates by mycotoxin production and aggressiveness to wheat

A total of 27Fusarium culmorum isolates from Germany and 41F. graminearum isolates from Kenya were investigated for aggressiveness and mycotoxin production on wheat ears. In addition, ergosterol content of the kernels from ears inoculated withF. graminearum was determined and theF. culmorum isolates were tested for mycotoxin productionin vitro. For both pathogens, isolates markedly differed in aggressiveness. 59% and 37% of theF. culmorum isolates produced NIV and DON, respectively,in vivo andin vitro. The DON-producing isolates also produced 3-acDONin vitro. The more aggressive isolates produced mainly DON while the less aggressive isolates produced mainly NIV. 12% and 85% of theF. graminearum isolates produced NIV and DON, respectively. The highly aggressive isolates produced higher amounts of DON, aggressiveness being highly correlated to DON content in the kernels. NIV-producing isolates were less aggressive. Ergosterol content of kernels was moderately correlated to aggressiveness but highly correlated to DON content. Disease severity was associated with kernel weight reduction.     

Release of overexpressed CypB activates ERK signaling through CD147 binding for hepatoma cell resistance to oxidative stress

Cyclophilin, a cytosolic receptor for the immunosuppressive drug cyclosporin A, plays a role in diverse pathophysiologies along with its receptor, CD147. Although the interaction between cyclophilin A and CD147 is well established in inflammatory disease, that of cyclophilin B (CypB) with CD147 has not been fully explored, especially in cancer cell biology, and the exact molecular mechanism underlying such an association is poorly understood. In this study, we first identified high expression levels of CypB in 54 % of hepatocellular carcinoma patient tissues but in only 12.5 % of normal liver tissues. Then, we demonstrated that CypB overexpression protects human hepatoma cells against oxidative stress through its binding to CD147; this protective effect depends on the peptidyl prolyl isomerase activity of CypB. siRNA-mediated knockdown of CypB expression rendered hepatoma cells more vulnerable to ROS-mediated apoptosis. Furthermore, we also determined that a direct interaction between secreted CypB and CD147 regulates the extracellular signal-regulated kinase intracellular signaling pathway and is indispensible for the protective functions of CypB. For the first time, we demonstrated that CypB has an essential function in protecting hepatoma cells against oxidative stress through binding to CD147 and regulating the ERK pathway.     

Mini Review: Immune Response to Myelin-Derived Sulfatide and CNS-Demyelination

Here we briefly review our understanding of the immune response to myelin-derived glycolipids during an inflammatory autoimmune response in the central nervous system (CNS). We focus primarily on the recognition of the self-glycolipid sulfatide by a distinct population of non-invariant NK T cells. The results of studies we have obtained so far in investigating the presentation of sulfatide by CNS-resident cells including microglia and their interactions with T cells indicate that this pathway might be successfully targeted for the treatment of autoimmune demyelination in multiple sclerosis. Special issue of the Journal dedicated to Drs. Anthony T. Campagnoni and Celia W. Campagnoni. It is a privilege to contribute an article for this special occasion. It was highly rewarding for me to participate in the Journal Club meetings at UCLA initiated by Tony in which experts in Neurochemistry, Neurosciences and Immunology would discuss and learn from each other with passion. I personally enjoyed and benefited both from these meetings and especially from the get-togethers at their lovely home overlooking the Santa Monica hills. I will always treasure my association and friendship with Tony and Celia.