Cerebroside sulfotransferase: preparation of antibody and localization of antigen in kidney

This immunohistochemical study describes the localization of the enzyme cerebroside sulfotransferase (phosphoadenosine phosphosulfate: galactosylceramide sulfotransferase, EC 2.8.2.11) in rat kidney. The enzyme was purified from kidney and the preparation was used to raise antibodies for immunocytochemical investigations. In the kidney, the antigen was present only on the brush border of the epithelial cells of the proximal tubules, suggesting that sulfation of glycolipids occurs in the cytoplasm and plasma membranes of these specific cells. Moreover, biochemical and immunocytochemical studies of cerebroside sulfotransferase during development indicate that catalytic activity is correlated with the appearance of enzyme protein.     

Tac antigen forms disulfide-linked homodimers

Interleukin 2 (IL-2) is responsible for stimulating T-cell proliferation via interaction with specific, high-affinity membrane receptors. Although an IL-2-binding protein has been identified by virtue of its reactivity with a monoclonal antibody that competes with IL-2 for binding (anti-Tac), the complete and precise structure of functional IL-2 receptors is still unknown. To define further the composition of IL-2 receptors, both IL-2 itself and anti-Tac were used as ligands to adsorb membrane proteins for analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (NaDodSO4/PAGE). A variety of experimental approaches yield results indicating that the Tac antigen, which migrates as a single protein on NaDod-SO4/PAGE under reducing conditions, is also expressed as disulfide-linked homodimers and oligomers. Examined under nonreducing conditions, both activated normal human T cells and cell lines from patients with adult T cell leukemia (HUT-102; MT-1) express Tac antigen homodimers (Mr 105,000) in addition to monomers (Mr 54,000). Formation of the disulfide bond is not a consequence of the experimental procedures used to isolate the proteins for analysis, inasmuch as identical results are obtained when the receptor proteins are iodinated and extracted in the presence of N-ethylmaleimide or prepared for electrophoresis in the absence of heat denaturation. Accordingly, these findings point to Tac antigen associating with itself preferentially. The physiologic significance of homodimer and oligomer formation, especially as it relates to the formation of high-affinity IL-2 receptors, is presently unknown.     

Cerebroside sulfotransferase deficiency ameliorates L-selectin-dependent monocyte infiltration in the kidney after ureteral obstruction

Mononuclear cells infiltrating the interstitium are involved in renal tubulointerstitial injury. The unilateral ureteral obstruction (UUO) is an established experimental model of renal interstitial inflammation. In our previous study, we postulated that L-selectin on monocytes is involved in their infiltration into the interstitium by UUO and that a sulfated glycolipid, sulfatide, is the physiological L-selectin ligand in the kidney. Here we tested the above hypothesis using sulfatide- and L-selectin-deficient mice. Sulfatide-deficient mice were generated by gene targeting of the cerebroside sulfotransferase (Cst) gene. Although the L-selectin-IgG chimera protein specifically bound to sulfatide fraction in acidic lipids from wild-type kidney, it did not show such binding in fractions of Cst(-/-) mice kidney, indicating that sulfatide is the major L-selectin-binding glycolipid in the kidney. The distribution of L-selectin ligand in wild-type mice changed after UUO; sulfatide was relocated from the distal tubules to the peritubular capillaries where monocytes infiltrate, suggesting that sulfatide relocated to the endothelium after UUO interacted with L-selectin on monocytes. In contrast, L-selectin ligand was not detected in Cst(-/-) mice irrespective of UUO treatment. Compared with wild-type mice, Cst(-/-) mice showed a considerable reduction in the number of monocytes/macrophages that infiltrated the interstitium after UUO. The number of monocytes/macrophages was also reduced to a similar extent in L-selectin(-/-) mice. Our results suggest that sulfatide is a major L-selectin-binding molecule in the kidney and that the interaction between L-selectin and sulfatide plays a critical role in monocyte infiltration into the kidney interstitium.     

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.     

Cerebroside sulfotransferase activity in human lung tissues. An elevated level in lung adenocarcinoma.

Cerebroside sulfotransferase activity was demonstrated in particulate fractions from human lung and its carcinoma tissues. The activity in human lung adenocarcinoma was significantly higher than those in a different histological type of carcinoma (squamous cell carcinoma) and in normal tissue from which each carcinoma was derived.     

Cytochrome b(5) forms homomeric complexes in living cells

Cytochrome b(5) (cyt-b(5)) is a ubiquitous hemoprotein also associated with microsomal cytochrome P450 17α-hydroxylase/17,20 lyase (CYP17A1). In the steroidogenic pathway CYP17A1 catalyses the metabolism of pregnenolone, yielding both glucocorticoid and androgen precursors. While not affecting the 17α-hydroxylation of pregnenolone, cyt-b(5) augments the 17,20 lyase reaction of 17-hydroxypregnenolone, catalyzing the formation of DHEA, through direct protein-protein interactions. In this study, multimeric complex formation of cyt-b(5) and the possible regulatory role of these complexes were investigated. Cyt-b(5) was isolated from ovine liver and used to raise anti-sheep cyt-b(5) immunoglobulins. Immunochemical studies revealed that, in vivo, cyt-b(5) is primarily found in the tetrameric form. Subsequent fluorescent resonance energy transfer (FRET) studies in COS-1 cells confirmed the formation of homomeric complexes by cyt-b(5) in live cells. Site-directed mutagenesis revealed that the C-terminal linker domain of cyt-b(5) is vital for complex formation. The 17,20-lyase activity of CYP17 was augmented by truncated cyt-b(5), which is unable to form complexes when co-expressed in COS-1 cells, thereby implicating the monomeric form of cyt-b(5) as the active species. This study has shown for the first time that cyt-b(5) forms homomeric complexes in vivo, implicating complex formation as a possible regulatory mechanism in steroidogenesis.     

The ToxR protein of Vibrio cholerae forms homodimers and heterodimers.

The ToxR protein of Vibrio cholerae regulates the expression of several virulence factors that play important roles in the pathogenesis of cholera. Previous experiments with ToxR-alkaline phosphatase (ToxR-PhoA) fusion proteins suggested a model for gene regulation in which the inactive form of ToxR was a monomer and the active form of ToxR was a dimer (V. L. Miller, R. K. Taylor, and J. J. Mekalanos, Cell 48:271-279, 1987). In order to examine whether ToxR exists in a dimeric form in vivo, biochemical cross-linking analyses were carried out. Different dimeric cross-linked species were detected depending on the expression level of ToxR: when overexpressed, ToxR+ToxR homodimers and ToxR+ToxS heterodimers were detected, and when ToxR was expressed at normal levels, exclusively ToxR+ToxS heterodimers were detected. The amount of overexpression was quantitated by using ToxR-PhoA fusion proteins and was found to correspond to 2.7-fold the normal level of ToxR. The formation of both homodimeric ToxR species and heterodimeric ToxR+ToxS species is consistent with previously reported genetic data that suggested that both types of ToxR oligomeric interactions occur. However, variation in the amount of either the homodimeric or heterodimeric form detectable by this cross-linking analysis was not observed to correlate with laboratory culture conditions known to modulate ToxR activity. Thus, genetic and biochemical data indicate that ToxR is able to interact with both itself and ToxS but that these interactions may not explain mechanistically the observed changes in ToxR activity that occur in response to environmental conditions.     

Representation of living forms

The living forms represented in this paper are sets of parts that spontaneously increase in organization. Their organizations are measured by an information-theoretic function derived from the work of Boltzmann and Shannon. We briefly review its derivation in the context of the troubled role of mathematics in biology, and then define the function. We illustrate its nature by measuring the 22 different organizations of a set of eight things; and we facilitate its use by defining the parameters that determine an amount of organization. The measure is then applied to show that the organization of limb pairs on free-living arthropods, based on data given by Cisne, confirms a pattern of increasing organization in their evolution from the Cambrian era to the present. Further applications measure the changes in organizations of ideal (theoretical) life forms, and contrasting changes in inanimate systems. Our main results represent the reproduction of unicellular organisms, and the formation of hierarchies, as processes of increasing organization.     

Su e of the yeast F1Fo-ATP synthase forms homodimers

The yeast F(1)F(o)-ATP synthase forms a dimeric complex in the mitochondrial inner membrane. Dimerization of two F(1)F(o) monomeric complexes involves the physical association of two membrane-embedded F(o) sectors and in a manner, which is dependent on the F(o) subunit, Su e. Sequence analysis of Su e protein family members indicated the presence of a conserved coiled-coil motif. As this motif is often the basis for protein homodimerization events, it was hypothesized that Su e forms homodimers in the inner membrane and that formation of Su e dimers between two neighboring F(o) complexes would facilitate dimerization of the F(1)F(o)-ATP synthase complex (Arnold, I., Pfeiffer, K., Neupert, W., Stuart, R. A., and Sch?gger, H. (1998) EMBO J. 17, 7170-7178). Using a histidine-tagged derivative of yeast Su e, Su e-His(12), combined with cross-linking and affinity purification approaches, we have directly demonstrated the ability of the yeast Su e protein to form homodimers. Functionality of the Su e-His(12) derivative was confirmed by its ability to assemble into the ATP synthase complex and to support its dimerization in the Deltasu e null mutant yeast cells. The close association of two neighboring Su e proteins was also demonstrated using cross-linking with Cu(2+), which binds and cross-links a unique Cys residue in neighboring Su e proteins. Finally, we propose a model for the molecular basis of the homodimerization of the Su e proteins.     

Biochemical and biophysical characterization of serotonin 5-HT2C receptor homodimers on the plasma membrane of living cells

While many studies have provided evidence of homodimerization and heterodimerization of G-protein-coupled receptors (GPCRs), few studies have used fluorescence resonance energy transfer (FRET) combined with confocal microscopy to visualize receptor dimerization on the plasma membrane, and there have been no reports demonstrating the expression of serotonin receptor dimers/oligomers on the plasma membrane of living cells. In the study presented here, biochemical and biophysical techniques were used to determine if 5-HT(2C) receptors exist as homodimers on the plasma membrane of living cells. Immunoprecipitation followed by Western blotting revealed the presence of immunoreactive bands the predicted size of 5-HT(2C) receptor monomers and homodimers that were detergent and cross-linker sensitive. Bioluminescence resonance energy transfer (BRET) was assessed in HEK293 cells expressing 5-HT(2C) receptors labeled with Renilla luciferase and yellow fluorescent protein. BRET levels were not altered by pretreatment with serotonin. Confocal microscopy provided direct visualization of FRET on the plasma membrane of live cells expressing 5-HT(2C) receptors labeled with cyan (donor) and yellow (acceptor) fluorescent proteins. FRET, assessed by acceptor photobleaching, was dependent on the donor/acceptor ratio and independent of acceptor expression levels, indicating that FRET resulted from receptor clustering and not from overexpression of randomly distributed receptors, providing evidence for GPCR dimers/oligomers in a clustered distribution on the plasma membrane. The results of this study suggest that 5-HT(2C) receptors exist as constitutive homodimers on the plasma membrane of living cells. In addition, a confocal-based FRET method for monitoring receptor dimerization directly on the plasma membrane of living cells is described.     

Kar2p availability defines distinct forms of endoplasmic reticulum stress in living cells

Accumulation of misfolded secretory proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) stress pathway. To enhance secretory protein folding and promote adaptation to stress, the UPR upregulates ER chaperone levels, including BiP. Here we describe chromosomal tagging of KAR2, the yeast homologue of BiP, with superfolder green fluorescent protein (sfGFP) to create a multifunctional endogenous reporter of the ER folding environment. Changes in Kar2p-sfGFP fluorescence levels directly correlate with UPR activity and represent a robust reporter for high-throughput analysis. A novel second feature of this reporter is that photobleaching microscopy (fluorescence recovery after photobleaching) of Kar2p-sfGFP mobility reports on the levels of unfolded secretory proteins in individual cells, independent of UPR status. Kar2p-sfGFP mobility decreases upon treatment with tunicamycin or dithiothreitol, consistent with increased levels of unfolded proteins and the incorporation of Kar2p-sfGFP into slower-diffusing complexes. During adaptation, we observe a significant lag between down-regulation of the UPR and resolution of the unfolded protein burden. Finally, we find that Kar2p-sfGFP mobility significantly increases upon inositol withdrawal, which also activates the UPR, apparently independent of unfolded protein levels. Thus Kar2p mobility represents a powerful new tool capable of distinguishing between the different mechanisms leading to UPR activation in living cells.     

Fluorescence and multiphoton imaging resolve unique structural forms of sterol in membranes of living cells

Although cholesterol is an essential component of mammalian membranes, resolution of cholesterol organization in membranes and organelles (i.e. lysosomes) of living cells is hampered by the paucity of nondestructive, nonperturbing methods providing real time structural information. Advantage was taken of the fact that the emission maxima of a naturally occurring fluorescent sterol (dehydroergosterol) were resolvable into two structural forms, monomeric (356 and 375 nm) and crystalline (403 and 426 nm). Model membranes (sterol:phospholipid ratios in the physiological range, e.g. 0.5-1.0), subcellular membrane fractions (plasma membranes, lysosomal membranes, microsomes, and mitochondrial membranes), and lipid rafts/caveolae (plasma membrane cholesterol-rich microdomain purified by a nondetergent method) contained primarily monomeric sterol and only small quantities (i.e. 1-5%) of the crystalline form. In contrast, the majority of sterol in isolated lysosomes was crystalline. However, addition of sterol carrier protein-2 in vitro significantly reduced the proportion of crystalline dehydroergosterol in the isolated lysosomes. Multiphoton laser scanning microscopy (MPLSM) of living L-cell fibroblasts cultured with dehydroergosterol for the first time provided real time images showing the presence of monomeric sterol in plasma membranes, as well as other intracellular membrane structures of living cells. Furthermore, MPLSM confirmed that crystalline sterol colocalized in highest amounts with LysoTracker Green, a lysosomal marker dye. Although crystalline sterol was also detected in the cytoplasm, the extralysosomal crystalline sterol did not colocalize with BODIPY FL C(5)-ceramide, a Golgi marker, and crystals were not associated with the cell surface membrane. These noninvasive, nonperturbing methods demonstrated for the first time that multiple structural forms of sterol normally occurred within membranes, membrane microdomains (lipid rafts/caveolae), and intracellular organelles of living cells, both in vitro and visualized in real time by MPLSM.     

The Escherichia coli cell division protein ZipA forms homodimers prior to association with FtsZ

ZipA is an essential component of the cell division machinery in E. coli and other closely related bacteria. It is an integral membrane protein that binds to FtsZ, tethering it to the inner membrane. ZipA also induces bundling of FtsZ protofilaments and may play a role in regulating FtsA activity; however, the molecular details behind these observations are not clear. In this study we have analyzed the oligomeric state of ZipA in vivo, by chemical cross-linking, and in vitro, by native gel electrophoresis (BN-PAGE). Our data indicate that ZipA can self-associate as a homodimer and that this self-interaction is not dependent on the FtsZ-binding domain. This observation rules out the possibility that FtsZ polymers mediate the ZipA self-interaction. Given this observation, it is possible that a certain population of ZipA is recruited to the division septum in a homodimeric form.     

Bcl-xL forms two distinct homodimers at non-ionic detergents: implications in the dimerization of Bcl-2 family proteins

As the key regulator of apoptosis, Bcl-2 family protein controls the cell death by forming homo- or heterodimers among anti-apoptotic and pro-apoptotic members of this family. Here we have studied Bcl-x(L) homodimerization at different pH in the presence of various detergents and organic solvents. We found that both acidic and basic pHs are beneficial for Bcl-x(L) dimerization. High concentrations of non-ionic detergents and some organic solvents can significantly promote this event. In addition to non-covalently linked acidic-dimer as that formed at acidic pH, Bcl-x(L) formed disulphide-bonded detergent-dimer at neutral and basic pH when incubated with high concentrations of non-ionic detergents. The acidic-dimer retains the BH3 peptide binding activity, whereas the detergent-dimer does not. The formation of acidic-dimer and detergent-dimer implies that Bcl-x(L) may dimerize via two different pathways under certain conditions. The implications of these findings has been discussed with previous experimental results, which provides some new insight into the events and would help the experiment design and data interpretation when non-ionic detergents are used to study the dimerization and pore formation of Bcl-2 family proteins.     

Shear stress mediates tyrosylprotein sulfotransferase isoform shift in human endothelial cells

In this study, we examined expression of tyrosylprotein sulfotransferase (TPST) isoforms TPST1 and TPST2 in primary cultures of human umbilical vein endothelial cells. For the first time coexpression of both isoforms is shown in primary human cells. Application of physiological levels of shear stress regulates expression of TPST isoforms in a time- and dose-dependent manner. Sustained application of arterial laminar shear stress causes downregulation of TPST1 mRNA and protein expression, while TPST2 is upregulated. This TPST isoform shift is mediated by different signaling pathways. Shear stress-dependent downregulation of TPST1 involves tyrosine kinase, while upregulation of TPST2 is mediated by a protein kinase C-dependent pathway [corrected].     

Estradiol-17beta sulfotransferase activity in canine osteosarcoma D17 cells

Estrogen sulfatase and sulfotransferase (EST) activities are present in breast cancer tissues but there are no reports on EST in cancerous bone cells. We incubated [(3)H]estradiol-17beta with cells from a canine osteosarcoma D17 line for periods up to 24 h. Radioactive steroids were recovered from the media and separated into unconjugated and conjugated fractions using Sep-Pak C18 cartridges. The conjugate fraction was solvolyzed and the resulting free steroids were obtained from a second C18 cartridge. Little metabolism was apparent in 4 h of incubation, but by 24 h as much as one half of the radioactivity was seen in the conjugate fraction. Most of the conjugates were recovered as sulfates in all three experiments. HPLC profiles showed a limited metabolism of estradiol to other compounds except for estrone, which was clearly present in both free and sulfate fractions. These results suggest that EST may have a role in the local metabolism of estrogens in bone.