Sugar-binding properties of VIP36, an intracellular animal lectin operating as a cargo receptor

The vesicular integral protein of 36 kDa (VIP36) is an intracellular animal lectin that acts as a putative cargo receptor, which recycles between the Golgi and the endoplasmic reticulum. Although it is known that VIP36 interacts with glycoproteins carrying high mannose-type oligosaccharides, detailed analyses of the sugar-binding specificity that discriminates isomeric oligosaccharide structures have not yet been performed. In the present study, we have analyzed, using the frontal affinity chromatography (FAC) method, the sugar-binding properties of a recombinant carbohydrate recognition domain of VIP36 (VIP36-CRD). For this purpose, a pyridylaminated sugar library, consisting of 21 kinds of oligosaccharides, including isomeric structures, was prepared and subjected to FAC analyses. The FAC data have shown that glucosylation and trimming of the D1 mannosyl branch interfere with the binding of VIP36-CRD. VIP36-CRD exhibits a bell-shaped pH dependence of sugar binding with an optimal pH value of approximately 6.5. By inspection of the specificity and optimal pH value of the sugar binding of VIP36 and its subcellular localization, together with the organellar pH, we suggest that VIP36 binds glycoproteins that retain the intact D1 mannosyl branch in the cis-Golgi network and recycles to the endoplasmic reticulum where, due to higher pH, it releases its cargos, thereby contributing to the quality control of glycoproteins.     

MDR-like ABC transporter AtPGP4 is involved in auxin-mediated lateral root and root hair development

Previous data have suggested an involvement of MDR/PGP-like ABC transporters in transport of the plant hormone auxin and, recently, AtPGP1 has been demonstrated to catalyze the primary active export of auxin. Here we show that related isoform AtPGP4 is expressed predominantly during early root development. AtPGP4 loss-of-function plants reveal enhanced lateral root initiation and root hair lengths both known to be under the control of auxin. Further, atpgp4 plants show altered sensitivities toward auxin and the auxin transport inhibitor, NPA. Finally, mutant roots reveal elevated free auxin levels and reduced auxin transport capacities. These results together with yeast growth assays suggest a direct involvement of AtPGP4 in auxin transport processes controlling lateral root and root hair development.     

Protease-sensitive signalling by chemically engineered intramolecular fluorescent resonance energy transfer mutants of green fluorescent protein

The native cysteine residues of green fluorescent protein (GFP) at positions 48 and 70 were replaced by non-thiolic amino acids, and new cysteine sites were introduced at specific, surface positions. Based on molecular modeling of the GFP structure, the sites chosen for mutagenesis to Cys were glutamic acid at position 6 and isoleucine at position 229. These new, unique cysteine sites provided reactive thiol groups suitable for site-specific chemical modification by eosin-based fluorescence labels. The new constructs were designed to serve as the basis of proof of principle for fluorescence resonance energy transfer (FRET) using an enzyme-activated (trypsin) intervening sequence between native and chemically conjugated fluorophores. These eosin moieties provided chemical FRET partners for the native GFP chromophore. On excitation, these GFP-eosin constructs exhibited strong intramolecular FRET, with quenching of the native GFP (511 nm) fluorophore emission and emission around 540 nm, corresponding to eosin. GFP mutants engineered with trypsin-sensitive sequences close to the eosin site, so that on trypsinolysis FRET was destroyed, the emission wavelength switching from that of the chemical FRET partner back to that of the native GFP fluorophore, providing efficient, ratio-based detection. This protein engineering provides the basis for novel bioprobes for enzymatic triggering using intramolecular FRET between GFP and carefully sited chemical labels.     

Discrete roles for histone acetylation in human T helper 1 cell-specific gene expression

To better understand the control of T helper (TH) 1-expressed genes, we compared and contrasted acetylation and expression for three key genes, IFNG, TBET, and IL18RAP and found them to be distinctly regulated. The TBET and the IFNG genes, but not the IL18RAP gene, showed preferential acetylation of histones H3 and H4 during TH1 differentiation. Analysis of acetylation of specific histone residues revealed that H3(Lys-9), H4(Lys-8), and H4(Lys-12) were preferentially modified in TH1 cells, suggesting a possible contribution of acetylation of these residues for induction of these genes. On the other hand, the acetylation of IL18RAP gene occurred both in TH1 and TH2 cells the similar kinetics and on the same with residues, demonstrating that selective histone acetylation was not universally the case for all TH1-expressed genes. Histone H3 acetylation of IFNG and TBET genes occurred with different kinetics, however, and was distinctively regulated by cytokines. Interleukin (IL)-12 and IL-18 enhanced the histone acetylation of the IFNG gene. By contrast, histone acetylation of the TBET gene was markedly suppressed by IL-4, whereas IL-12 and IL-18 had only modest effects suggesting that histone acetylation during TH1 differentiation is a process that is regulated by various factors at multiple levels. By treating Th2 cells with a histone deacetylase inhibitor, we restored histone acetylation of the IFNG and TBET genes, but it did not fully restore their expression in TH2 cells, again suggesting that histone acetylation explains one but not all the aspects of TH1-specific gene expression.     

UV light-induced DNA damage and tolerance for the survival of nucleotide excision repair-deficient human cells

DNA damage can cause cell death unless it is either repaired or tolerated. The precise contributions of repair and tolerance mechanisms to cell survival have not been previously evaluated. Here we have analyzed the cell killing effect of the two major UV light-induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs), in nucleotide excision repair-deficient human cells by expressing photolyase(s) for light-dependent photorepair of either or both lesions. Immediate repair of the less abundant 6-4PPs enhances the survival rate to a similar extent as the immediate repair of CPDs, indicating that a single 6-4PP lesion is severalfold more toxic than a CPD in the cells. Because UV light-induced DNA damage is not repaired at all in nucleotide excision repair-deficient cells, proliferation of these cells after UV light irradiation must be achieved by tolerance of the damage at replication. We found that RNA interference designed to suppress polymerase zeta activity made the cells more sensitive to UV light. This increase in sensitivity was prevented by photorepair of 6-4PPs but not by photorepair of CPDs, indicating that polymerase zeta is involved in the tolerance of 6-4PPs in human cells.     

Inhibition of hepatocyte growth factor induction in human dermal fibroblasts by interleukin-1 and its prevention by interferon-gamma

Hepatocyte growth factor (HGF) is one of the vital factors for liver regeneration. HGF production is induced by the activation of protein kinase A and protein kinase C-mediated pathways, interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and epidermal growth factor (EGF) in mesenchymal cells. We here report that IL-1 and TNF-alpha, hitherto regarded as HGF inducers, potently inhibited HGF production stimulated by other HGF inducers. IL-1alpha, IL-1beta, and TNF-alpha alone had minimal stimulating effects on HGF production in human dermal fibroblasts, but they strongly inhibited production of HGF induced by cholera toxin, 8-bromo-cAMP, EGF, and phorbol 12-myristate 13-acetate (PMA). Moreover, although the high level of HGF production in MRC-5 cells was enhanced by PMA and less markedly by IL-1beta, HGF production in MRC-5 cells treated with PMA plus IL-1beta was less than that in the cells treated with PMA alone. In the presence of interferon (IFN)-gamma, however, cholera toxin- and 8-bromo-cAMP-induced HGF production was not inhibited by IL-1beta. Pretreatment of cells with IL-1beta suppressed the phosphorylation of cAMP-responsive element-binding protein induced by cholera toxin but not that induced by 8-bromo-cAMP. Taken together, our results indicate that IL-1 inhibited HGF production stimulated by various inducers, including protein kinase A-activating agents, and that IFN-gamma overcame this inhibition of induction of HGF production.     

MAIGO2 is involved in exit of seed storage proteins from the endoplasmic reticulum in Arabidopsis thaliana

Seed storage proteins are synthesized on the endoplasmic reticulum (ER) as precursors and then transported to protein storage vacuoles, where they are processed into mature forms. Here, we isolated an Arabidopsis thaliana mutant, maigo2 (mag2), that accumulated the precursors of two major storage proteins, 2S albumin and 12S globulin, in dry seeds. mag2 seed cells contained many novel structures, with an electron-dense core that was composed of the precursor forms of 2S albumin. 12S globulins were segregated from 2S albumin and were localized in the matrix region of the structures together with the ER chaperones lumenal binding protein and protein disulfide isomerase, which were more abundant in mag2 seeds. The MAG2 gene was identified as At3g47700, and the MAG2 protein had a RINT-1/TIP20 domain in the C-terminal region. We found that some MAG2 molecules were peripherally associated with the ER membrane. MAG2 had an ability to bind to two ER-localized t-SNAREs (for target-soluble NSF [N-ethylmaleimide-sensitive fusion protein] attachment protein receptor; At Sec20 and At Ufe1). Our findings suggest that MAG2 functions in the transport of storage protein precursors between the ER and Golgi complex in plants.     

Preparative fractionation of protein, RNA, and plasmid DNA using centrifugal precipitation chromatography with tubular dialysis membrane inside a convoluted tubing as separation channel

Fractionation of clarified E. coli lysate components in bench-scale and preparative-scale centrifugal precipitation chromatography (CPC), using a solution of cationic surfactant cetyltrimethylammonium bromide (CTAB) containing 0.5 M NaCl as precipitant, are compared here. Step gradient of CTAB from 0.50% to 0.16% (w/v) gave a successful fractionation in bench-scale CPC; however, a linear gradient of lower CTAB concentration, 0.20-0% (w/v), was used in the preparative scale and resulted in similar fractionation. The preparative-scale CPC has a superior sample loading capacity by the use of tubular dialysis membrane inside convoluted tubing as the separation channel. In this study, the quantity of the sample loaded into the preparative CPC was about 15 times more than that in the bench scale, and in a single run the preparative CPC could prepare approximately 3 mg of plasmid DNA with about 96% of RNA removed. The higher surface area per length of the separation channel in the preparative CPC was believed to benefit mass transfer of CTAB across the membrane, leading to less CTAB being required in the process.     

Noradrenaline stimulates ATP release from DRG neurons by targeting β3 adrenoceptors as a factor of neuropathic pain

Noradrenaline (NA), released in association with sympathetic nerve sprouting into the dorsal root ganglion (DRG) after peripheral nerve injury, may enhance neuropathic pain. ATP serves as a pain mediator; however, NA‐regulated ATP mobilizations in the DRG is far from understanding. In the present study, we analyzed ATP mobilizations in acutely dissociated rat DRG neurons by recording single‐channel currents through P2X receptor channels as an ATP biosensor in an outside‐out patch‐clamp configuration and by monitoring real‐time enzymatic NADPH fluorescent imaging, and examined the role for β₃ adrenoceptors in allodynia using an in vivo rat model. We show here that NA stimulates ATP release from DRG neurons as mediated via β₃ adrenoceptors linked to G_s protein involving PKA activation, to cause allodynia. This represents a fresh regulatory pathway for neuropathic pain relevant to noradrenergic transmission in the DRG. J. Cell. Physiol. 224: 345–351, 2010. © 2010 Wiley‐Liss, Inc.     

Apoptotic cells activate NKT cells through T cell Ig-like mucin-like-1 resulting in airway hyperreactivity

T cell Ig-like mucin-like-1 (TIM-1) is an important asthma susceptibility gene, but the immunological mechanisms by which TIM-1 functions remain uncertain. TIM-1 is also a receptor for phosphatidylserine (PtdSer), an important marker of cells undergoing programmed cell death, or apoptosis. We now demonstrate that NKT cells constitutively express TIM-1 and become activated by apoptotic cells expressing PtdSer. TIM-1 recognition of PtdSer induced NKT cell activation, proliferation, and cytokine production. Moreover, the induction of apoptosis in airway epithelial cells activated pulmonary NKT cells and unexpectedly resulted in airway hyperreactivity, a cardinal feature of asthma, in an NKT cell-dependent and TIM-1-dependent fashion. These results suggest that TIM-1 serves as a pattern recognition receptor on NKT cells that senses PtdSer on apoptotic cells as a damage-associated molecular pattern. Furthermore, these results provide evidence for a novel innate pathway that results in airway hyperreactivity and may help to explain how TIM-1 and NKT cells regulate asthma.