Inactivation of parkin by oxidative stress and C-terminal truncations: a protective role of molecular chaperones

Loss of parkin function is linked to autosomal recessive juvenile parkinsonism. Here we show that proteotoxic stress and short C-terminal truncations induce misfolding of parkin. As a consequence, wild-type parkin was depleted from a high molecular weight complex and inactivated by aggregation. Similarly, the pathogenic parkin mutant W453Stop, characterized by a C-terminal deletion of 13 amino acids, spontaneously adopted a misfolded conformation. Mutational analysis indicated that C-terminal truncations exceeding 3 amino acids abolished formation of detergent-soluble parkin. In the cytosol scattered aggregates of misfolded parkin contained the molecular chaperone Hsp70. Moreover, increased expression of chaperones prevented aggregation of wild-type parkin and promoted folding of the W453Stop mutant. Analyzing parkin folding in vitro indicated that parkin is aggregation-prone and that its folding is dependent on chaperones. Our study demonstrates that C-terminal truncations impede parkin folding and reveal a new mechanism for inactivation of parkin.     

HDL derived from the different phases of conjugated diene formation reduces membrane fluidity and contributes to a decrease in free cholesterol efflux from human THP-1 macrophages

Oxidized HDL (ox-HDL) has been reported to reduce free cholesterol efflux from cells. In this study we investigate the effect of different stages of ox-HDL on macrophage membrane fluidity and its effect on free cholesterol efflux from macrophages as a cell function influenced by ox-HDL. HDL was oxidized by means of conjugated diene production using copper as a prooxidant. Fluidity of HDL and human THP-1 macrophage membranes was evaluated by changes in fluorescence anisotropy (r) by DPH probe where lower (r) values give higher fluidity. We found that ox-HDL derived from the propagation phase (PP-HDL) and the decomposition phase (DP-HDL) became less fluid ((r): 0.263+/-0.001, 0.279+/-0.002, respectively) than HDL from the lag phase (LP-HDL) and native HDL (nat-HDL) ((r): 0.206+/-0.001) (P<0.05). Macrophages incubated with PP-HDL and DP-HDL had less fluid membranes ((r): 0.231+/-0.001, 0.243+/-0.002, respectively) than those incubated with LP-HDL and nat-HDL ((r): 0.223+/-0.001) (P<0.05). Consequently, fluidity was reduced not only in ox-HDL but also in the cell membranes exposed to ox-HDL. A significant negative correlation was observed between macrophage membrane fluorescence anisotropy (r) and free cholesterol efflux from these cells (-0.876; P<0.05). Thus, lower membrane fluidity was associated with lower free cholesterol efflux from cells. In conclusion, the increase in the HDL oxidation process leads to a lost of macrophage membrane fluidity that could contribute to an explanation of the reduction of free cholesterol efflux from cells by ox-HDL.     

RhoA exerts a permissive effect on volume-regulated anion channels in vascular endothelial cells

Cell swelling triggers in most cell typesan outwardly rectifying anion current,I_Cl,swell, via volume-regulated anion channels (VRACs). We have previously demonstrated in calf pulmonary artery endothelial (CPAE) cells that inhibition of the Rho/Rho kinase/myosin light chain phosphorylation pathway reduces the swelling-dependent activation of I_Cl,swell. However, theseexperiments did not allow us to discriminate between a direct activatorrole or a permissive effect. We now show that the Rho pathway did notaffect VRAC activity if this pathway was activated by transfecting CPAEcells with constitutively active isoforms of G (a Rho activatingheterotrimeric G protein subunit), Rho, or Rho kinase. Furthermore,biochemical and morphological analysis failed to demonstrate activationof the Rho pathway during hypotonic cell swelling. Finally,manipulating the Rho pathway with either guanosine5'-O-(3-thiotriphosphate) or C3 exoenzyme had no effect onVRACs in caveolin-1-expressing Caco-2 cells. We conclude that the Rhopathway exerts a permissive effect on VRACs in CPAE cells, i.e.,swelling-induced opening of VRACs requires a functional Rho pathway,but not an activation of the Rho pathway.

     

The matrix metalloproteinase 9 (mmp-9) hemopexin domain is a novel gelatin binding domain and acts as an antagonist

Matrix metalloproteinases (MMPs) are involved in the remodeling processes of the extracellular matrix and the basement membrane. Most MMPs are composed of a regulatory, a catalytic, and a hemopexin subunit. In many tumors the expression of MMP-9 correlates with local tumor growth, invasion, and metastasis. To analyze the role of the hemopexin domain in these processes, the MMP-9 hemopexin domain (MMP-9-PEX) was expressed as a glutathione S-transferase fusion protein in Escherichia coli. After proteolytic cleavage, the isolated PEX domain was purified by size exclusion chromatography. In a zymography assay, MMP-9-PEX was able to inhibit MMP-9 activity. The association and dissociation rates for the interaction of MMP-9-PEX with gelatin were determined by plasmon resonance. From the measured rate constants, the dissociation constant was calculated to be K(d) = 2,4 x 10(-8) m, demonstrating a high affinity between MMP-9-PEX and gelatin. In Boyden chamber experiments the recombinant MMP-9-PEX was able to inhibit the invasion of melanoma cells secreting high amounts of MMP-9 in a dose-dependent manner. These data demonstrate for the first time that the hemopexin domain of MMP-9 has a high affinity binding site for gelatin, and the particular recombinant domain is able to block MMP-9 activity and tumor cell invasion. Because MMP-9 plays an important role in metastasis, this antagonistic effect may be utilized to design MMP inhibition-based cancer therapy.     

Orientational constraints of the gp130 intracellular juxtamembrane domain for signaling

The glycoprotein 130 (gp130) is the common signal transducing receptor chain of the interleukin-6 family of cytokines. Here we investigated the requirements for transfer of the information given by ligand binding to the cytoplasmic domain of gp130. It is demonstrated that the box 1/2 region has to be located membrane-proximally in order to bind and activate Janus kinases. To test the possible requirement of an alpha-helical orientation, we inserted 1-4 alanine residues into this juxtamembrane intracellular region. The insertion of one alanine results in a strongly reduced activation of STAT1 and STAT3, whereas insertion of three alanine residues leads to a stronger STAT activation. These results suggest that gp130-mediated activation of STATs is sensitive to rotational changes around the receptor axis perpendicular to the membrane. Surprisingly, insertion of 1, 2, 3, or 4 alanine residues into this juxtamembrane region leads to successive impairment but not abolishment of Janus kinase and receptor phosphorylation, supporting the finding of sensitivity of Janus kinases toward changes in distance of box 1/2 from the plasma membrane. We suggest a new model concerning the gp130 activation mode in which the relative orientation of the cytoplasmic regions seems to be critical for further signal transduction.     

Glutamine synthetase enhances the clearance of extracellular glutamate by the neural retina

Clearance of synaptic glutamate by glial cells is required for the normal function of excitatory synapses and for prevention of neurotoxicity. Although the regulatory role of glial glutamate transporters in glutamate clearance is well established, little is known about the influence of glial glutamate metabolism on this process. This study examines whether glutamine synthetase (GS), a glial-specific enzyme that amidates glutamate to glutamine, affects the uptake of glutamate. Retinal explants were incubated in the presence of [(14)C]glutamate and glutamate uptake was assessed by measurement of the amount of radioactively labeled molecules within the cells and the amount of [(14)C]glutamine released to the medium. An increase in GS expression in Müller glial cells, caused by induction of the endogenous gene, did not affect the amount of glutamate accumulated within the cells, but led to a dramatic increase in the amount of glutamine released. This increase, which was directly correlated with the level of GS expression, was dependent on the presence of external sodium ions, and could be completely abolished by methionine sulfoximine, a specific inhibitor of GS activity. Our results demonstrate that GS activity significantly influences the uptake of glutamate by the neural retina and suggest that this enzyme may represent an important target for neuroprotective strategies.     

Jasmonate is involved in the induction of tyrosine aminotransferase and tocopherol biosynthesis in Arabidopsis thaliana

Coronatine-inducible tyrosine aminotransferase (TAT), which catalyses the transamination from tyrosine to p-hydroxyphenylpyruvate, is the first enzyme of a pathway leading via homogentisic acid to plastoquinone and tocopherols, the latter of which are known to be radical scavengers in plants. TAT can be also induced by the octadecanoids methyl jasmonate (MeJA) and methyl-12-oxophytodienoic acid (MeOPDA), as well as by wounding, high light, UV light and the herbicide oxyfluorfen. In order to elucidate the role of octadecanoids in the process of TAT induction in Arabidopsis thaliana (L.) Heynh., the jasmonate-deficient mutant delayed dehiscence (dde1) was used, in which the gene for 12-oxophytodienoic acid reductase 3 is disrupted. The amount of immunodetectable TAT was low. The enzyme was still fully induced by coronatine as well as by MeJA although induction by the latter was to a lesser extent and later than in the wild type. Treatment with MeOPDA, wounding and UV light, however, had hardly any effects. Tocopherol levels that showed considerable increases in the wild type after some treatments were much less affected in the mutant. However, starting levels of tocopherol were higher in non-induced dde1 than in the wild type. We conclude that jasmonate plays an important role in the signal transduction pathway regulating TAT activity and the biosynthesis of its product tocopherol.     

Differential control of vesicle priming and short-term plasticity by Munc13 isoforms

Presynaptic short-term plasticity is an important adaptive mechanism regulating synaptic transmitter release at varying action potential frequencies. However, the underlying molecular mechanisms are unknown. We examined genetically defined and functionally unique axonal subpopulations of synapses in excitatory hippocampal neurons that utilize either Munc13-1 or Munc13-2 as synaptic vesicle priming factor. In contrast to Munc13-1-dependent synapses, Munc13-2-driven synapses show pronounced and transient augmentation of synaptic amplitudes following high-frequency stimulation. This augmentation is caused by a Ca(2+)-dependent increase in release probability and releasable vesicle pool size, and requires phospholipase C activity. Thus, differential expression of Munc13 isoforms at individual synapses represents a general mechanism that controls short-term plasticity and contributes to the heterogeneity of synaptic information coding.