A new member of the protein kinase C family, nPKC theta, predominantly expressed in skeletal muscle.

A new protein kinase C (PKC)-related cDNA with unique tissue distribution has been isolated and characterized. This cDNA encodes a protein, nPKC theta, which consists of 707 amino acid residues and showed the highest sequence similarity to nPKC delta (67.0% in total). nPKC theta has a zinc-finger-like cysteine-rich sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are common in all PKC family members. However, nPKC theta lacks a putative Ca2+ binding region (C2 region) that is seen only in the conventional PKC subfamily (cPKC alpha, -beta I, -beta II, and -gamma) but not in the novel PKC subfamily (nPKC delta, -epsilon, -zeta, and -eta). Northern (RNA) blot analyses revealed that the mRNA for nPKC theta is expressed predominantly in skeletal muscle. Furthermore, nPKC theta mRNA is the most abundantly expressed PKC isoform in skeletal muscle among the nine PKC family members. nPKC theta expressed in COS1 cells serves as a phorbol ester receptor. By the use of an antipeptide antibody specific to the D2-D3 region of the nPKC theta sequence, nPKC theta was recognized as a 79-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in mouse skeletal muscle extract and also in an extract from COS1 cells transfected with an nPKC theta cDNA expression plasmid. Autophosphorylation of immunoprecipitated nPKC theta was observed; it was enhanced by phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate but attenuated by the addition of Ca2+. These results clearly demonstrate that nPKC theta should be considered a member of the PKC family of proteins that play crucial roles in the signal transduction pathway.     

Occurrence and subcellular location of NADH- and NADPH-linked aquacobalamin reductases in human liver

1. Both activities of NADH- and NADPH-linked aquacobalamin reductases were found in some human tissues, liver, kidney pancreas, heart, spleen, lung, cerebrum, cerebellum, adrenal glands, stomach, duodenum, jejunum, ileum, colon and bone marrow. 2. Human liver contained both enzymes with higher specific activities than any other tissues. 3. The liver NADH-linked enzyme was distributed in both mitochondrial (approx. 60%) and microsomal (40%) fractions; similar to the distribution of the NADPH-linked enzyme, but of which 40% activity was found in the mitochondria and the remaining activity was recovered in the microsomes. 4. The results suggest that the synthetic systems of the cobalamin coenzymes occur in both mitochondria and microsomes of human liver.     

Site-specific phosphorylation by protein kinase C inhibits assembly-promoting activity of microtubule-associated protein 4

We have examined the phosphorylation of bovine microtubule-associated protein 4 (MAP4), formerly named MAP-U, by protein kinase C (PKC). When MAP4 was incubated with PKC, about 1 mol of phosphate was incorporated/mol of MAP4. Phosphorylation of MAP4 caused a remarkable decrease in the ability of the MAP to stimulate microtubule assembly. MAP4 consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain. The carboxyl-terminal domain is subdivided into a Pro-rich region and an assembly-promoting (AP) sequence region containing four tandem repeats of AP sequence that is conserved in MAP4, MAP2, and tau [Aizawa et al. (1990) J. Biol. Chem. 265, 13849-13855]. In order to identify the site of MAP4 phosphorylated by PKC, a series of expressed MAP4 fragments was prepared and treated with the kinase. A fragment corresponding to the Pro-rich region (P fragment) was phosphorylated, while fragments corresponding to the projection domain and the AP sequence region were not. In addition, chymotryptic digestion of an authentic MAP4 prephosphorylated by PKC revealed that phosphate was incorporated almost exclusively into a 27-kDa fragment containing the carboxyl-terminal half of the Pro-rich region. We investigated the phosphorylation site in MAP4 using the P fragment and found that Ser815 was phosphorylated almost exclusively. We conclude that the phosphorylation of a single Ser residue in the Pro-rich region negatively regulates the assembly-promoting activity of MAP4.     

A point mutation in ribosomal protein L7/L12 reduces its ability to form a compact dimer structure and to assemble into the GTPase center

An Escherichia coli mutant, LL103, harboring a mutation (Ser15 to Phe) in ribosomal protein L7/L12 was isolated among revertants of a streptomycin-dependent strain. In the crystal structure of the L7/L12 dimer, residue 15 within the N-terminal domain contacts the C-terminal domain of the partner monomer. We tested effects of the mutation on molecular assembly by biochemical approaches. Gel electrophoretic analysis showed that the Phe15-L7/L12 variant had reduced ability in binding to L10, an effect enhanced in the presence of 0.05% of nonionic detergent. Mobility of Phe15-L7/L12 on gel containing the detergent was very low compared to the wild-type proteins, presumably because of an extended structural state of the mutant L7/L12. Ribosomes isolated from LL103 cells contained a reduced amount of L7/L12 and showed low levels (15-30% of wild-type ribosomes) of activities dependent on elongation factors and in translation of natural mRNA. The ribosomal activity was completely recovered by addition of an excess amount of Phe15-L7/L12 to the ribosomes, suggesting that the mutant L7/L12 exerts normal functions when bound on the ribosome. The interaction of Ser15 with the C-terminal domain of the partner molecule seems to contribute to formation of the compact dimer structure and its efficient assembly into the ribosomal GTPase center. We propose a model relating compact and elongated forms of L7/L12 dimers. Phe15-L7/L12 provides a new tool for studying the functional structure of the homodimer.     

BACE1 interacts with nicastrin

Beta-amyloid peptide (Abeta) is generated through the proteolytic cleavage of beta-amyloid precursor protein (APP) by beta- and gamma-secretases. The beta-secretase, BACE1, initiates Abeta formation followed by gamma-cleavage within the APP transmembrane domain. Although BACE1 localizes in the transGolgi network (TGN), its physiological substrates and modulators are not known. In addition, the relationship to other secretase(s) also remains unidentified. Here, we demonstrate that BACE1 binds to nicastrin, a component of gamma-secretase complexes, in vitro, and that nicastrin activates beta-secretase activity in COS-7 cells.     

The crucial role of caspase-9 in the disease progression of a transgenic ALS mouse model

Mutant copper/zinc superoxide dismutase (SOD1)-overexpressing transgenic mice, a mouse model for familial amyotrophic lateral sclerosis (ALS), provides an excellent resource for developing novel therapies for ALS. Several observations suggest that mitochondria-dependent apoptotic signaling, including caspase-9 activation, may play an important role in mutant SOD1-related neurodegeneration. To elucidate the role of caspase-9 in ALS, we examined the effects of an inhibitor of X chromosome-linked inhibitor of apoptosis (XIAP), a mammalian inhibitor of caspase-3, -7 and -9, and p35, a baculoviral broad caspase inhibitor that does not inhibit caspase-9. When expressed in spinal motor neurons of mutant SOD1 mice using transgenic techniques, XIAP attenuated disease progression without delaying onset. In contrast, p35 delayed onset without slowing disease progression. Moreover, caspase-9 was activated in spinal motor neurons of human ALS subjects. These data strongly suggest that caspase-9 plays a crucial role in disease progression of ALS and constitutes a promising therapeutic target.     

Metabolism of amyloid precursor protein in COS cells transfected with a β-secretase candidate

Thimet oligopeptidase (TOP) is a thiol- andmetallo-dependent peptidase and has been shown to beone of the -secretase candidates. TOPexpressed in COS cells cleaved amyloid precursorprotein (APP) at the -secretase site, and wefound a proteolytic product of APP called secretedform of APP by -secretase (sAPP) in theconditioned media. Here we demonstrate thatsAPP was increased in conditioned media whenTOP was coexpressed in COS cells with APP and treatedwith an ADAM inhibitor SI-27. In addition, althoughTOP expressed in COS cell was localized at nuclei orGolgi apparatus, it exclusively colocalized at Golgiapparatus when APP was coexpressed with TOP.     

Novel Pathway for Conversion of Chlorohydroxyquinol to Maleylacetate in Burkholderia cepacia AC1100

Burkholderia cepacia AC1100 metabolizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) via formation of 5-chlorohydroxyquinol (5-CHQ), hydroxyquinol (HQ), maleylacetate, and β-oxoadipate. The step(s) leading to the dechlorination of 5-CHQ to HQ has remained unidentified. We demonstrate that a dechlorinating enzyme, TftG, catalyzes the conversion of 5-CHQ to hydroxybenzoquinone, which is then reduced to HQ by a hydroxybenzoquinone reductase (HBQ reductase). HQ is subsequently converted to maleylacetate by hydroxyquinol 1,2-dioxygenase (HQDO). All three enzymes were purified. We demonstrate specific product formation by colorimetric assay and mass spectrometry when 5-CHQ is treated successively with the three enzymes: TftG, TftG plus HBQ reductase, and TftG plus HBQ reductase plus HQDO. This study delineates the complete enzymatic pathway for the degradation of 5-CHQ to maleylacetate.     

Presenilin 1 Mutations Linked to Familial Alzheimer's Disease Increase the Intracellular Levels of Amyloid β-Protein 1–42 and Its N-Terminally Truncated Variant(s) Which Are Generated at Distinct Sites

Abstract: Mutations in the presenilin genes PS1 and PS2 cause the most common form of early-onset familial Alzheimer's disease. The influence of PS1 mutations on the generation of endogenous intracellular amyloid β-protein (Aβ) species was assessed using a highly sensitive immunoblotting technique with inducible mouse neuro-blastoma (Neuro 2a) cell lines expressing the human wild-type (wt) or mutated PS1 (M146L or Δexon 10). The induction of mutated PS1 increased the intracellular levels of two distinct Aβ species ending at residue 42 that were likely to be Aβ1–42 and its N-terminally truncated variant(s) Aβx-42. The induction of mutated PS1 resulted in a higher level of intracellular Aβ1–42 than of intracellular Aβx-42, whereas extracellular levels of Aβ1–42 and Aβx-42 were increased proportionally. In addition, the intracellular generation of these Aβ42 species in wt and mutated PS1 -induced cells was completely blocked by brefeldin A, whereas it exhibited differential sensitivities to monensin: the increased accumulation of intracellular Aβx-42 versus inhibition of intracellular Aβ1–42 generation. These data strongly suggest that Aβx-42 is generated in a proximal Golgi, whereas Aβ1–42 is generated in a distal Golgi and/or a post-Golgi compartment. Thus, it appears that PS1 mutations enhance the degree of 42-specific γ-secretase cleavage that occurs in the normal β-amyloid precursor protein processing pathway (a) in the endoplasmic reticulum or the early Golgi apparatus prior to β-secretase cleavage or (b) in the distinct sites where Aβx-42 and Aβ1–42 are generated.