Search

The regulatory isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a light-activated enzyme constituted by subunits GapA and GapB. The NADPH-dependent activity of regulatory GAPDH from spinach chloroplasts was affected by the redox potential (E(m,7.9), -353 +/- 11 mV) through the action of thioredoxin f. The redox dependence of recombinant GapB (E(m,7.9), -347 +/- 9 mV) was similar to native GAPDH, whereas GapA was essentially redox-insensitive. GapB mutants having one or two C-terminal cysteines mutated into serines (C358S, C349S, C349S/C358S) were less redox-sensitive than GapB. Different mutants with other cysteines substituted by serines (C18S, C274S, C285S) still showed strong redox regulation. Fully active GapB was a tetramer of B-subunits, and, when incubated with NAD, it associated to a high molecular weight oligomer showing low NADPH-dependent activity. The C-terminal GapB mutants (C358S, C349S, C349S/C358S) were active tetramers unable to aggregate to higher oligomers in the presence of NAD, whereas other mutants (C18S, C274S, C285S) again behaved like GapB. We conclude that a regulatory disulfide, between Cys-349 and Cys-358 of the C-terminal extension of GapB, does form in the presence of oxidized thioredoxin. This covalent modification is required for the NAD-dependent association into higher oligomers and inhibition of the NADPH-activity. By leading to GAPDH autoinhibition, thioredoxin and NAD may thus concur to the dark inactivation of the enzyme in vivo.

The importance of voltage-activated calcium channels in pain processing has been suggested by the spinal antinociceptive action of blockers of N- and P/Q-type calcium channels as well as by gene targeting of the alpha1B subunit (N-type). The accessory beta3 subunits of calcium channels are preferentially associated with the alpha1B subunit in neurones. Here we show that deletion of the beta3 subunit by gene targeting affects strongly the pain processing of mutant mice. We pinpoint this defect in the pain-related behavior and ascending pain pathways of the spinal cord in vivo and at the level of calcium channel currents and proteins in single dorsal root ganglion neurones in vitro. The pain induced by chemical inflammation is preferentially damped by deletion of beta3 subunits, whereas responses to acute thermal and mechanical harmful stimuli are reduced moderately or not at all, respectively. The defect results in a weak wind-up of spinal cord activity during intense afferent nerve stimulation. The molecular mechanism responsible for the phenotype was traced to low expression of N-type calcium channels (alpha1B) and functional alterations of calcium channel currents in neurones projecting to the spinal cord.

Interstitial cells of Cajal (ICC) are considered to be pacemaker cells in gastrointestinal tracts. ICC generate electrical rhythmicity (dihydropyridine-insensitive) as slow waves and drive spontaneous contraction of smooth muscles. Although cytosolic Ca2+has been assumed to play a key role in pacemaking, Ca2+movements in ICC have not yet been examined in detail. In the present study, using cultured cell clusters isolated from mouse small intestine, we demonstrated Ca2+oscillations in ICC. Fluo-4 was loaded to the cell cluster, the relative amount of cytosolic Ca2+was recorded, and ICC were identified by c-Kit immunoreactivity. We specifically detected Ca2+oscillation in ICC in the presence of dihydropyridine, which abolishes Ca2+oscillation in smooth muscles. The oscillation was coupled to the electrical activity corresponding to slow waves, and it depended on Ca2+influx through a non-selective cation channel, which was SK&F 96365-sensitive and store-operated. We further demonstrated the presence of transient receptor potential-like channel 4 (TRP4) in caveolae of ICC. Taken together, the results infer that the Ca2+oscillation in ICC is intimately linked to the pacemaker function and depends on Ca2+influx mediated by TRP4.

The regulation of intracellular Ca2+plays a key role in the development and growth of cells. Here we report the cloning and functional expression of a highly calcium-selective channel localized on the human chromosome 7. The sequence of the new channel is structurally related to the gene product of the CaT1 protein cloned from rat duodenum and is therefore called CaT-like (CaT-L). CaT-L is expressed in locally advanced prostate cancer, metastatic and androgen-insensitive prostatic lesions but is undetectable in healthy prostate tissue and benign prostatic hyperplasia. Additionally, CaT-L is expressed in normal placenta, exocrine pancreas, and salivary glands. New markers with well defined biological function that correlate with aberrant cell growth are needed for the molecular staging of cancer and to predict the clinical outcome. The human CaT-L channel represents a marker for prostate cancer progression and may serve as a target for therapeutic strategies.

Mammalian homologues of DrosophilaTrp have been implicated to form channels that are activated following the depletion of Ca2+from internal stores. Recent studies indicate that actin redistribution is required for the activation of these channels. Here we show that murine Trp4 and Trp5, as well as phospholipase C β1 and β2 interact with the first PDZ domain of NHERF, regulatory factor of the Na+/H+exchanger. We demonstrated the association of Trp4 and phospholipase C-β1 with NHERF in vivoin an HEK293 cell line expressing Trp4 and in adult mouse brain by immuno-coprecipitation. NHERF is a two PDZ domain-containing protein that associates with the actin cytoskeleton via interactions with members of ezrin/radixin/moesin family. Thus, store-operated channels involving Trp4 and Trp5 can form signaling complexes with phospholipase C isozymes via interactions with NHERF and thereby linking the lipase and the channels to the actin cytoskeleton. The interaction with the PDZ protein may constitute an important mechanism for distribution and regulation of store-operated channels.

Mammalian TRP proteins have been implicated to function as ion channel subunits responsible for agonist-induced Ca2+entry. To date, TRP proteins have been extensively studied by heterologous expression giving rise to diverse channel properties and activation mechanisms including store-operated mechanisms. However, the molecular structure and the functional properties of nativeTRP channels still remain elusive. Here we analyze the properties of TRP4 (CCE1) channels in their native environment and characterize TRP expression patterns and store-operated calcium currents that are endogenous to bovine adrenal cells. We show by Northern blot analysis, immunoblots, and immunohistochemistry thatTRP4transcripts and TRP4 protein are present in the adrenal cortex but absent in the medulla. Correspondingly, bovine adrenal cortex cells express TRP4 abundantly. The only otherTRPtranscript found at considerable levels wasTRP1, whereas TRP2, TRP3, TRP5(CCE2),and TRP6were not detectable. Depletion of calcium stores with inositol 1,4,5-trisphosphate or thapsigargin activates store-operated ion channels in adrenal cells. These channels closely resemble calcium release-activated Ca2+(CRAC) channels. Expression of trp4(CCE1)cDNA in antisense orientation significantly reduces both, the endogenous CRAC-like currents and the amount of native TRP4 protein. These results demonstrate that TRP4 contributes essentially to the formation of native CRAC-like channels in adrenal cells.

In skeletal muscle the oligomeric alpha(1S), alpha(2)/delta-1 or alpha(2)/delta-2, beta1, and gamma1 L-type Ca(2+) channel or dihydropyridine receptor functions as a voltage sensor for excitation contraction coupling and is responsible for the L-type Ca(2+) current. The gamma1 subunit, which is tightly associated with this Ca(2+) channel, is a membrane-spanning protein exclusively expressed in skeletal muscle. Previously, heterologous expression studies revealed that gamma1 might modulate Ca(2+) currents expressed by the pore subunit found in heart, alpha(1C), shifting steady state inactivation, and increasing current amplitude. To determine the role of gamma1 assembled with the skeletal subunit composition in vivo, we used gene targeting to establish a mouse model, in which gamma1 expression is eliminated. Comparing litter-matched mice with control mice, we found that, in contrast to heterologous expression studies, the loss of gamma1 significantly increased the amplitude of peak dihydropyridine-sensitive I(Ca) in isolated myotubes. Whereas the activation kinetics of the current remained unchanged, inactivation of the current was slowed in gamma1-deficient myotubes and, correspondingly, steady state inactivation of I(Ca) was shifted to more positive membrane potentials. These results indicate that gamma1 decreases the amount of Ca(2+) entry during stimulation of skeletal muscle.

In skeletal muscle the oligomeric α1S, α2/δ-1 or α2/δ-2, β1, and γ1 L-type Ca2+channel or dihydropyridine receptor functions as a voltage sensor for excitation contraction coupling and is responsible for the L-type Ca2+current. The γ1 subunit, which is tightly associated with this Ca2+channel, is a membrane-spanning protein exclusively expressed in skeletal muscle. Previously, heterologous expression studies revealed that γ1 might modulate Ca2+currents expressed by the pore subunit found in heart, α1C, shifting steady state inactivation, and increasing current amplitude. To determine the role of γ1 assembled with the skeletal subunit composition in vivo, we used gene targeting to establish a mouse model, in which γ1 expression is eliminated. Comparing litter-matched mice with control mice, we found that, in contrast to heterologous expression studies, the loss of γ1 significantly increased the amplitude of peak dihydropyridine-sensitive ICain isolated myotubes. Whereas the activation kinetics of the current remained unchanged, inactivation of the current was slowed in γ1-deficient myotubes and, correspondingly, steady state inactivation of ICawas shifted to more positive membrane potentials. These results indicate that γ1 decreases the amount of Ca2+entry during stimulation of skeletal muscle.

A novel protein kinase, Mx-interacting protein kinase (PKM), has been identified in a yeast two-hybrid screen for interaction partners of human MxA, an interferon-induced GTPase with antiviral activity against several RNA viruses. A highly conserved protein kinase domain is present in the N-terminal moiety of PKM, whereas an Mx interaction domain overlaps with C-terminal PEST sequences. PKM has a molecular weight of about 127,000 and exhibits high sequence homology to members of a recently described family of homeodomain-interacting protein kinases. Recombinant PKM has serine/threonine kinase activity that is abolished by a single amino acid substitution in the ATP binding domain (K221W). PKM catalyzes autophosphorylation and phosphorylation of various cellular and viral proteins. PKM is expressed constitutively and colocalizes with the interferon-inducible Sp100 protein and murine Mx1 in discrete nuclear structures known as nuclear bodies.

Polypeptide D1 of the photosystem II reaction center of oxygenic photosynthesis is expressed in precursor form (pre-D1), and it must be proteolytically processed at its C terminus to enable assembly of the manganese cluster responsible for photosynthetic water oxidation. A rapid and highly sensitive enzyme-linked immunosorbent assay-based microtiter plate method is described for assaying this D1 C-terminal processing protease. A protocol is described for the isolation and purification to homogeneity of the enzyme from the green alga, Scenedesmus obliquus. Amino acid sequence information on the purified protease was used to clone the corresponding gene, the translated sequence of which is presented. A comparison of the gene product with homologous proteases points to a region of conserved residues that likely corresponds to the active site of a new class of serine protease. The LF-1 mutant strain of Scenedesmus (isolated by Dr. Norman Bishop) is incapable of processing pre-D1. We show here that the C-terminal processing protease gene in this strain contains a single base deletion that causes a frame shift and a premature stop of translation within the likely active site of the enzyme. A suppressor strain, LF-1-RVT-1, which is photoautotrophic and capable of processing pre-D1 has a nearby single base insertion that restores the expression of active enzyme. These observations provide the first definitive proof that the enzyme isolated is responsible for in vivo proteolytic processing of pre-D1 and that no other protease can compensate for its loss.

Polypeptide D1 of the photosystem II reaction center of oxygenic photosynthesis is expressed in precursor form (pre-D1), and it must be proteolytically processed at its C terminus to enable assembly of the manganese cluster responsible for photosynthetic water oxidation. A rapid and highly sensitive enzyme-linked immunosorbent assay-based microtiter plate method is described for assaying this D1 C-terminal processing protease. A protocol is described for the isolation and purification to homogeneity of the enzyme from the green alga, Scenedesmus obliquus. Amino acid sequence information on the purified protease was used to clone the corresponding gene, the translated sequence of which is presented. A comparison of the gene product with homologous proteases points to a region of conserved residues that likely corresponds to the active site of a new class of serine protease. The LF-1 mutant strain ofScenedesmus(isolated by Dr. Norman Bishop) is incapable of processing pre-D1. We show here that the C-terminal processing protease gene in this strain contains a single base deletion that causes a frame shift and a premature stop of translation within the likely active site of the enzyme. A suppressor strain, LF-1-RVT-1, which is photoautotrophic and capable of processing pre-D1 has a nearby single base insertion that restores the expression of active enzyme. These observations provide the first definitive proof that the enzyme isolated is responsible for in vivoproteolytic processing of pre-D1 and that no other protease can compensate for its loss.