Common MCL1 polymorphisms associated with risk of tuberculosis

MCL1 expression has been found to be up-regulated during infection with virulent Mycobacterium tuberculosis. We investigated the genetic polymorphisms in MCL1 as potential candidate gene for a host genetic study of clinical TB infection. We have sequenced exons and their boundaries of MCL1, including the 1.5 kb promoter region, to identify polymorphisms, and eight polymorphisms were identified. The genetic associations of polymorphisms in MCL1 with clinical TB patients (n=486) and normal controls (n=370) were analyzed. Using statistical analyses, one common promoter polymorphism (MCL1- 324C > A) which is absolutely linked with three other SNPs in the promoter and 3'UTR regions, were found to be significantly associated with increased risk of clinical TB disease. The frequency of the A-bearing genotype of -324C > A was higher in clinical TB patients than in normal controls (P=0.0008, OR= 1.68). Our findings suggest that polymorphisms in MCL1 might be one of genetic factors for the risk of clinical tuberculosis development.     

Dynein light chain LC8 negatively regulates NF-kappaB through the redox-dependent interaction with IkappaBalpha

Redox regulation of nuclear factor kappaB (NF-kappaB) has been described, but the molecular mechanism underlying such regulation has remained unclear. We recently showed that a novel disulfide reductase, TRP14, inhibits tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation, and we identified the dynein light chain LC8, which interacts with the NF-kappaB inhibitor IkappaBalpha, as a potential substrate of TRP14. We now show the molecular mechanism by which NF-kappaB activation is redox-dependently regulated through LC8. LC8 inhibited TNFalpha-induced NF-kappaB activation in HeLa cells by interacting with IkappaBalpha and thereby preventing its phosphorylation by IkappaB kinase (IKK), without affecting the activity of IKK itself. TNFalpha induced the production of reactive oxygen species, which oxidized LC8 to a homodimer linked by the reversible formation of a disulfide bond between the Cys(2) residues of each subunit and thereby resulted in its dissociation from IkappaBalpha. Butylated hydroxyanisol, an antioxidant, and diphenyleneiodonium, an inhibitor of NADPH oxidase, attenuated the phosphorylation and degradation of IkappaBalpha by TNFalpha stimulation. In addition LC8 inhibited NF-kappaB activation by other stimuli including interleukin-1beta and lipopolysaccharide, both of which generated reactive oxygen species. Furthermore, TRP14 catalyzed reduction of oxidized LC8. Together, our results indicate that LC8 binds IkappaBalpha in a redox-dependent manner and thereby prevents its phosphorylation by IKK. TRP14 contributes to this inhibitory activity by maintaining LC8 in a reduced state.     

Regulation of MAPK phosphatase 1 (AtMKP1) by calmodulin in Arabidopsis

The mitogen-activated protein kinases (MAPKs) are key signal transduction molecules, which respond to various external stimuli. The MAPK phosphatases (MKPs) are known to be negative regulators of MAPKs in eukaryotes. We screened an Arabidopsis cDNA library using horseradish peroxidase-conjugated calmodulin (CaM), and isolated AtMKP1 as a CaM-binding protein. Recently, tobacco NtMKP1 and rice OsMKP1, two orthologs of Arabidopsis AtMKP1, were reported to bind CaM via a single putative CaM binding domain (CaMBD). However, little is known about the regulation of phosphatase activity of plant MKP1s by CaM binding. In this study, we identified two Ca(2+)-dependent CaMBDs within AtMKP1. Specific binding of CaM to two different CaMBDs was verified using a gel mobility shift assay, a competition assay with a Ca(2+)/CaM-dependent enzyme, and a split-ubiquitin assay. The peptides for two CaMBDs, CaMBDI and CaMBDII, bound CaM in a Ca(2+)-dependent manner, and the binding affinity of CaMBDII was found to be higher than that of CaMBDI. CaM overlay assays using mutated CaMBDs showed that four amino acids, Trp(453) and Leu(456) in CaMBDI and Trp(678) and Ile(684) in CaMBDII, play a pivotal role in CaM binding. Moreover, the phosphatase activity of AtMKP1 was increased by CaM in a Ca(2+)-dependent manner. Our results suggest that two important signaling pathways, Ca(2+) signaling and the MAPK signaling cascade, are connected in plants via the regulation of AtMKP1 activity. To our knowledge, this is the first report to show that the biochemical activity of MKP1 in plants is regulated by CaM.     

Regulation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) by Src involves tyrosine phosphorylation of PDK1 and Src homology 2 domain binding

3-Phosphoinositide-dependent protein kinase-1 (PDK1) appears to play a central regulatory role in many cell signalings between phosphoinositide-3 kinase and various intracellular serine/threonine kinases. In resting cells, PDK1 is known to be constitutively active and is further activated by tyrosine phosphorylation (Tyr(9) and Tyr(373/376)) following the treatment of the cell with insulin or pervanadate. However, little is known about the mechanisms for this additional activation of PDK1. Here, we report that the SH2 domain of Src, Crk, and GAP recognized tyrosine-phosphorylated PDK1 in vitro. Destabilization of PDK1 induced by geldanamycin (a Hsp90 inhibitor) was partially blocked in HEK 293 cells expressing PDK1-Y9F. Co-expression of Hsp90 enhanced PDK1-Src complex formation and led to further increased PDK1 activity toward PKB and SGK. Immunohistochemical analysis with anti-phospho-Tyr(9) antibodies showed that the level of Tyr(9) phosphorylation was markedly increased in tumor samples compared with normal. Taken together, these data suggest that phosphorylation of PDK1 on Tyr(9), distinct from Tyr(373/376), is important for PDK1/Src complex formation, leading to PDK1 activation. Furthermore, Tyr(9) phosphorylation is critical for the stabilization of both PDK1 and the PDK1/Src complex via Hsp90-mediated protection of PDK1 degradation.     

Crystal structure of Escherichia coli MazG, the regulator of nutritional stress response

MazG is a nucleoside triphosphate pyrophosphohydrolase that hydrolyzes all canonical nucleoside triphosphates. The mazG gene located downstream from the chromosomal mazEF "addiction module," that mediated programmed cell death in Escherichia coli. MazG activity is inhibited by the MazEF complex both in vivo and in vitro. Enzymatic activity of MazG in vivo affects the cellular level of guanosine 3',5'-bispyrophosphate (ppGpp), synthesized by RelA under amino acid starvation. The reduction of ppGpp, caused by MazG, may extend the period of cell survival under nutritional stress. Here we describe the first crystal structure of active MazG from E. coli, which is composed of two similarly folded globular domains in tandem. Among the two putative catalytic domains, only the C-terminal domain has well ordered active sites and exhibits an NTPase activity. The MazG-ATP complex structure and subsequent mutagenesis studies explain the peculiar active site environment accommodating all eight canonical NTPs as substrates. In vivo nutrient starvation experiments show that the C terminus NTPase activity is responsible for the regulation of bacterial cell survival under nutritional stress.     

The outer segment serves as a default destination for the trafficking of membrane proteins in photoreceptors

Photoreceptors are compartmentalized neurons in which all proteins responsible for evoking visual signals are confined to the outer segment. Yet, the mechanisms responsible for establishing and maintaining photoreceptor compartmentalization are poorly understood. Here we investigated the targeting of two related membrane proteins, R9AP and syntaxin 3, one residing within and the other excluded from the outer segment. Surprisingly, we have found that only syntaxin 3 has targeting information encoded in its sequence and its removal redirects this protein to the outer segment. Furthermore, proteins residing in the endoplasmic reticulum and mitochondria were similarly redirected to the outer segment after removing their targeting signals. This reveals a pattern where membrane proteins lacking specific targeting information are delivered to the outer segment, which is likely to reflect the enormous appetite of this organelle for new material necessitated by its constant renewal. This also implies that every protein residing outside the outer segment must have a means to avoid this “default” trafficking flow.     

Etoposide-resistant HT-29 human colon carcinoma cells during glucose deprivation are sensitive to piericidin A, a GRP78 down-regulator

Glucose deprivation, a pathophysiological cell condition, causes up-regulation of GRP78 and induction of etoposide resistance in human cancer cells. The induction of drug resistance can be partly explained by the fact that GRP78 can block activation of caspase-7 induced by treatment with etoposide. Therefore, downregulating GRP78 expression may be a novel strategy anticancer drug development. Based on that premise, we established a screening program for anticancer agents that exhibit preferential cytotoxic activity for etoposide-resistant cancer cells under glucose-deprived conditions. We recently isolated an active compound, AR-054, from the culture broth of Streptomyces sp., which prevents stress-induced etoposide resistance in vitro. AR-054 was identified as piericidin A, a prototypical compound, by ESI-MS analysis and various NMR spectroscopic methods. Here, we showed that piericidin A suppressed the accumulation of GRP78 protein and was also highly toxic to etoposide-resistant HT-29 cells, with IC50 values for colony formation of 6.4 and 7.7 nM under 2-deoxyglucose supplemented and glucose-deprived conditions, respectively. Interestingly, piericidin A had no effect under normal growth conditions. Therefore, we suggest that piericidin A prevents up-regulation of GRP78, and exhibits cytotoxicity in glucose-deprived HT-29 cells that are resistant to etoposide.     

Synergistic effect of high glucose and ANG II on proliferation of mouse embryonic stem cells: involvement of PKC and MAPKs as well as AT1 receptor

This study examined the synergistic effect of high glucose levels and ANG II on proliferation and its related signal pathways using mouse embryonic stem (ES) cells. The combined use of a high glucose concentration (25 mM) and ANG II increased the level of [3H]thymidine/BrdU incorporation, and the number of cells compared with either treatment alone. Each treatment with high glucose or ANG II increased the cell population in the S phase compared with control, and the combined treatment of a high glucose concentration and ANG II significantly increased the number of cells in the S phase according to FACS analysis. Moreover, the high glucose-induced increase in [3H]thymidine incorporation was blocked by inhibiting the ANG II type 1 (AT1) receptor. The combined high glucose and ANG II significantly increased the STAT3 phosphorylation compared with high glucose or ANG II alone. ANG II stimulated the influx of Ca2+ in 25 mM glucose compared with 5 mM glucose. High glucose levels increase the level of PKC alpha, epsilon, and zeta translocation from the cytosol to the membrane fraction. In an examination of other signal pathways, the combined treatment significantly increased the level of p44/42, p38 MAPKs phosphorylation compared with either treatment alone. Indeed, the combined treatment increased the mRNA expression level of the protooncogenes and cell cycle regulatory proteins. In conclusion, the combined treatment of a high glucose concentration and ANG II had a synergistic effect in stimulating mouse ES cell proliferation through the Ca2+/PKC, MAPKs, and the AT1 receptor.     

Bis deficiency results in early lethality with metabolic deterioration and involution of spleen and thymus

Bcl-2 interacting cell death suppressor (Bis), also known as Bag3 or CAIR-1, is involved in antistress and antiapoptotic pathways. In addition to Bcl-2, Bis binds to several proteins, suggesting it has diverse functions in normal and pathological conditions. To better define the physiological function of Bis in vivo, we developed bis-deficient mice with a cre-loxP system. Targeted disruption of exon 4 of the bis gene was demonstrated by Southern blotting and PCR, and Western blotting showed that no intact or truncated Bis protein was synthesized in bis(-/-) mice. While heterozygotes were fertile and appeared normal, Bis-deficient mice showed growth retardation and died by 3 wk after birth. The relative weight of the thymus and spleen was reduced and the total numbers of white blood cells, splenocytes, and thymocytes were significantly reduced compared with wild-type littermates. Serum profiles indicated significant hypoglycemia as well as decrease in triglyceride and cholesterol levels. Expression profiles of metabolic genes indicated that gluconeogenesis and beta-oxidation are activated in the liver of bis(-/-) mice. This activation, as well as a decrease in peripheral fat and an induction of fatty liver, appears to be an adaptive response to hypoglycemia. Our study reveals that the absence of Bis has considerable influences on postnatal growth and survival, possibly due to a nutritional impairment.     

Regulation of stretch-activated ANP secretion by chloride channels

This study was aimed to define roles of stretch-activated ion channels (SACs), especially Cl(-) channels, in regulation of atrial natriuretic peptide (ANP) secretion using isolated perfused beating atria. The volume load was achieved by elevating height of outflow catheter connected to isolated rat atria and the pressure load was achieved by decreasing diameter of outflow catheter. Both methods increased atrial contractility similarly although volume load was different (736microl for volume load vs. 129microl for pressure load). Atrial stretch by volume load markedly increased ECF translocation and ANP secretion but the pressure load slightly increased. The ANP secretion was positively correlated to workload generated by volume or pressure load. Treatment of atria with gadolinium, a blocker for SACs, attenuated the ECF translocation and the ANP secretion induced by volume load. A blocker for Ca2+-activated Cl(-) channel, niflumic acid (NFA), accentuated the ANP secretion induced by volume load whereas a blocker for swelling-activated Cl(-) channel, diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), attenuated the ANP secretion. The ANP secretion of hypertrophied atria by volume load was markedly reduced and the augmented effect of NFA on volume load-induced ANP secretion was not observed. These results indicate that Cl(-) channels may differently regulate stretch-activated ANP secretion.