Protein kinase C nu/protein kinase D3 nuclear localization,catalytic activation,and intracellular redistribution in response to G protein-coupled receptor agonists

The protein kinase D (PKD) family consists of three serine/threonine kinases: PKC micro/PKD, PKD2, and PKCnu/PKD3. Whereas PKD has been the focus of most studies, virtually nothing is known about the effect of G protein-coupled receptor agonists (GPCR) on the regulatory properties and intracellular distribution of PKD3. Consequently, we examined the mechanism that mediates its activation and intracellular distribution. GPCR agonists induced a rapid activation of PKD3 by a protein kinase C (PKC)-dependent pathway that leads to the phosphorylation of the activation loop of PKD3. Comparison of the steady-state distribution of endogenous or tagged PKD3 versus PKD and PKD2 in unstimulated cells indicated that whereas PKD and PKD2 are predominantly cytoplasmic, PKD3 is present both in the nucleus and cytoplasm. This distribution of PKD3 results from its continuous shuttling between both compartments by a mechanism that requires a nuclear import receptor and a competent CRM1-nuclear export pathway. Cell stimulation with the GPCR agonist neurotensin induced a rapid and reversible plasma membrane translocation of PKD3 that is PKC-dependent. Interestingly, the nuclear accumulation of PKD3 can be dramatically enhanced in response to its activation. Thus, this study demonstrates that the intracellular distribution of PKD isoenzymes are distinct, and suggests that their signaling properties are regulated by differential localization.     

Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase

Efficient replication of hepatitis C virus (HCV) replicons in cell culture is associated with specific sequences not generally observed in vivo. These cell culture adaptive mutations dramatically increase the frequency with which replication is established in vitro. However, replicons derived from HCV isolates that have been shown to replicate in chimpanzees do not replicate in cell culture even when these adaptive mutations are introduced. To better understand this apparent paradox, we performed a gain-of-function screen to identify sequences that could confer cell culture replication competence to replicons derived from chimpanzee infectious HCV isolates. We found that residue 470 in domain II of the NS3 helicase is a critical determinant in cell culture adaptation. Substitutions in residue 470 when combined with the NS5A-S232I adaptive mutation are both necessary and sufficient to confer cell culture replication to otherwise inactive replicons, including those derived from genotype 1b HCV-BK and genotype 1a HCV-H77 isolates. The specific substitution at residue 470 required for replication is context-dependent, with R470M and P470L being optimal for the activity of HCV-BK and HCV-H77 replicons, respectively. Together these data indicate that mutations in the NS3 helicase domain II act in concert with previously identified adaptive mutations and predict that introduction of compatible residues at these positions can confer cell culture replication activity to diverse HCV isolates.     

One electron oxidation of benzyltrialkylsilanes catalysed by lignin peroxidase: comparison with the oxidation induced by chemical oxidants

Lignin peroxidase catalyses the H(2)O(2)-induced oxidation of 4-methoxybenzyltrimethylsilane by an electron transfer mechanism. The intermediate radical cation undergoes preferentially C(alpha)[bond]H deprotonation to give 4-methoxybenzaldehyde whereas C(alpha)[bond]Si bond cleavage is a minor fragmentation pathway and leads to 4-methoxybenzyl alcohol. Similar results are obtained in the oxidation catalysed by the water soluble model compound 5,10,15,20-tetra(N-methyl-4-pyridyl)porphyrinatoiron(III) pentachloride. Instead, in the oxidation promoted by the genuine one-electron transfer oxidant potassium dodecatungstocobalt(III)ate C(alpha)[bond]Si bond cleavage is the exclusive fragmentation process of the intermediate radical cation. It is suggested that in the enzymatic and biomimetic oxidations of 4-methoxybenzyltrimethylsilane the deprotonation of the intermediate radical cation is promoted by the reduced form [PorFe(IV)[double bond]O] of the active oxidant, which is an iron-oxo porphyrin radical cation.     

Intracellular redistribution of protein kinase D2 in response to G-protein-coupled receptor agonists

The protein kinase D (PKD) family consists of three serine/threonine protein kinases: PKC mu/PKD, PKD2, and PKC nu/PKD3. While PKD has been the focus of most studies to date, no information is available on the intracellular distribution of PKD2. Consequently, we examined the mechanism that regulates its intracellular distribution in human pancreatic carcinoma Panc-1 cells. Analysis of the intracellular steady-state distribution of fluorescent-tagged PKD2 in unstimulated cells indicated that this kinase is predominantly cytoplasmic. Cell stimulation with the G protein-coupled receptor agonist neurotensin induced a rapid and reversible plasma membrane translocation of PKD2 by a mechanism that requires PKC activity. In contrast to the other PKD isoenzymes, PKD2 activation did not induce its redistribution from the cytoplasm to the nucleus. Thus, this study demonstrates that the regulation of the distribution of PKD2 is distinct from other PKD isoenzymes, and suggests that the differential spatio-temporal localization of these signaling molecules regulates their specific signaling properties.     

Association of vitamin D receptor polymorphisms with osteoporosis in mexican postmenopausal women

It has been reported that Vitamin D receptor polymorphisms are associated with osteoporosis, particularly those demonstrated by the BsmI and FokI restriction enzymes. Herein we report the results of a case-control study performed in postmenopausal Mexican women. We studied 65 osteoporotic women (< or = -2.5 SD bone mineral density [BMD] of young normal females) and 57 controls (over 90% > or = -1.5 SD BMD of young normal females. Restriction enzymes BsmI and FokI were used to identify polymorphisms. Odds ratios and their 95% confidence intervals were calculated, and analysis was performed controlling for age as a covariate. The BsmI genotypes revealed a higher frequency of the bb genotype in cases than in controls, contradicting much of the literature that suggests this genotype protects females against osteoporosis. Regarding the FokI genotypes, we were unable to confirm that the FF genotype has a protective effect against osteoporosis. The inconsistencies found in the literature and the results obtained in the present work suggest to us that other genetic and nongenetic factors are involved in the occurrence of osteoporosis, confounding the results of the possible association of osteoporosis and VDR polymorphisms.     

Extracellular matrix remodeling and metalloproteinase involvement during intestine regeneration in the sea cucumber Holothuria glaberrima

The sea cucumber, Holothuria glaberrima, has the capacity to regenerate its internal organs. Intestinal regeneration is accomplished by the thickening of the mesenteric border and the invasion of this thickening by mucosal epithelium from the esophagus and the cloaca. Extracellular matrix (ECM) remodeling has been associated with morphogenetic events during embryonic development and regeneration. We have used immunohistochemical techniques against ECM components to show that differential changes occur in the ECM during early regeneration. Labeling of fibrous collagenous components and muscle-related laminin disappear from the regenerating intestine and mesentery, while fibronectin labeling and 4G7 (an echinoderm ECM component) are continuously present. Western blots confirm a decrease in fibrous collagen content during the first 2 weeks of regeneration. We have also identified five 1,10-phenanthroline-sensitive bands in collagen gelatin zymographs. The gelatinolytic activities of these bands are enhanced during early stages of regeneration, suggesting that the metalloprotease activity is associated with ECM remodeling. Inhibition of MMPs in vivo with 1,10-phenanthroline, p-aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamate or N-CBZ-Pro-Leu-Gly hydroxamate produces a reversible inhibition of intestinal regeneration and ECM remodeling. Our results show that significant changes in ECM content occur during intestine regeneration in the sea cucumber and that the onset of these changes is correlated to the proteolytic activities of MMPs.     

Disruption of sensitization to methylphenidate by a single administration of MK-801

Blockade of sensitization to methylphenidate by a single injection of MK-801 was investigated using a computerized activity monitoring system. Male Sprague-Dawley rats were housed in test cages and motor activity was recorded continuously for 16 days. After 2 days of baseline recording and a saline injection on day 3, the rats were randomly divided into four experimental groups. All received 2.5 mg/kg of methylphenidate (s.c.) once a day from days 4 to 9, then after five days of no treatment, they were re-challenged with 2.5 mg/kg of methylphenidate on day 15. One group received only methylphenidate, while the other three groups also received a single i.p. injection of MK-801 (0.30 mg/kg) either 24 h (day 3) or 1 h prior to the first of the six methylphenidate injections (day 4), or 1 h prior to the second methylphenidate injection (day 5). A single injection of MK-801 on day 4 (1 h prior to methylphenidate) blocked the development of sensitization to methylphenidate, since a sensitized response could not be elicited six days after cessation of repeated methylphenidate administration (day 15). However, sensitization to methylphenidate still occurred in the groups receiving MK-801 (0.30 mg/kg) on day 5, indicating that the mechanism by which a single injection of MK-801 disrupts sensitization to methylphenidate is sensitive to timing and is not a direct long-term effect. In conclusion, a single injection of MK-801 persistently blocks the development of sensitization to methylphenidate only if it is given with methylphenidate on the first day of the repetitive treatment phase.     

The protease core of the muscle-specific calpain,p94, undergoes Ca2+-dependent intramolecular autolysis

Limb girdle muscular dystrophy type 2A is linked to a skeletal muscle-specific calpain isoform known as p94. Isolation of the intact 94-kDa enzyme has been difficult to achieve due to its rapid autolysis, and uncertainty has arisen over its Ca2+-dependence for activity. We have expressed a C-terminally truncated form of the enzyme that comprises the protease core (domains I and II) along with its insertion sequence, IS1, and N-terminal leader sequence, NS. This 47-kDa p94I-II mini-calpain was stable during purification. In the presence of Ca2+, p94I-II cleaved itself within the NS and IS1 sequences. Mapping of the autolysis sites showed that NS and IS1 have the potential to be removed without damage to the protease core. Ca2+-dependent autolysis must be an intramolecular event because the inactive p94I-II C129S mutant was not cleaved by incubation with wild-type p94I-II. In addition, the rate of autolysis of p94I-II was independent of the concentration of the enzyme.     

Direct NMR observation and DFT calculations of a hydrogen bond at the active site of a 44 kDa enzyme

A hydrogen bond between the amide backbone of Arg7 and the remote imidazole side chain of His106 has been directly observed by improved TROSY-NMR techniques in the 44 kDa trimeric enzyme chorismate mutase from Bacillus subtilis. The presence of this hydrogen bond in the free enzyme and its complexes with a transition state analog and the reaction product was demonstrated by measurement of ¹⁵N-¹⁵N and ¹H-¹⁵N trans-hydrogen bond scalar couplings, ^2h J _NN and ^1h J _HN, and by transfer of nuclear polarization across the hydrogen bond. The conformational dependences of these coupling constants were analyzed using sum-over-states density functional perturbation theory (SOS-DFPT). The observed hydrogen bond might stabilize the scaffold at the active site of BsCM. Because the Arg7-His106 hydrogen bond has not been observed in any of the high resolution crystal structures of BsCM, the measured coupling constants provide unique information about the enzyme and its complexes that should prove useful for structural refinement of atomic models.