Comparative proteomic analysis of Methanothermobacter themautotrophicus ΔH in pure culture and in co-culture with a butyrate-oxidizing bacterium

To understand the physiological basis of methanogenic archaea living on interspecies H(2) transfer, the protein expression of a hydrogenotrophic methanogen, Methanothermobacter thermautotrophicus strain ΔH, was investigated in both pure culture and syntrophic coculture with an anaerobic butyrate oxidizer Syntrophothermus lipocalidus strain TGB-C1 as an H(2) supplier. Comparative proteomic analysis showed that global protein expression of methanogen cells in the model coculture was substantially different from that of pure cultured cells. In brief, in syntrophic coculture, although methanogenesis-driven energy generation appeared to be maintained by shifting the pathway to the alternative methyl coenzyme M reductase isozyme I and cofactor F(420)-dependent process, the machinery proteins involved in carbon fixation, amino acid synthesis, and RNA/DNA metabolisms tended to be down-regulated, indicating restrained cell growth rather than vigorous proliferation. In addition, our proteome analysis revealed that α subunits of proteasome were differentially acetylated between the two culture conditions. Since the relevant modification has been suspected to regulate proteolytic activity of the proteasome, the global protein turnover rate could be controlled under syntrophic growth conditions. To our knowledge, the present study is the first report on N-acetylation of proteasome subunits in methanogenic archaea. These results clearly indicated that physiological adaptation of hydrogenotrophic methanogens to syntrophic growth is more complicated than that of hitherto proposed.     

The E3 ubiquitin ligase activity of Trip12 is essential for mouse embryogenesis

Protein ubiquitination is a post-translational protein modification that regulates many biological conditions. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12(mt/mt)) that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12(mt/mt) embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16. In contrast, Trip12(mt/mt) ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12(mt/mt) ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex) and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development.     

Inhibition of neuronal nitric-oxide synthase by phosphorylation at Threonine1296 in NG108-15 neuronal cells

We demonstrate that neuronal nitric-oxide synthase (nNOS) is directly inhibited through the phosphorylation of Thr(1296) in NG108-15 neuronal cells. Treatment of NG108-15 cells expressing nNOS with calyculin A, an inhibitor of protein phosphatase 1 and 2A, revealed a dose-dependent inhibition of nNOS enzyme activity with concomitant phosphorylation of Thr(1296) residue. Cells expressing a phosphorylation-deficient mutant in which Thr(1296) was changed to Ala proved resistant to phosphorylation and suppression of NOS activity. Mimicking phosphorylation mutant of nNOS in which Thr(1296) is changed to Asp showed a significant decrease in nNOS enzyme activity, being competitive with NADPH, relative to the wild-type enzyme. These data suggest that phosphorylation of nNOS at Thr(1296) may involve the attenuation of nitric oxide production in neuronal cells through the decrease of NADPH-binding to the enzyme.     

Dynamic function of the alkyl spacer of acetogenins in their inhibitory action with mitochondrial complex I (NADH-ubiquinone oxidoreductase)

Studies on the inhibitory mechanism of acetogenins, the most potent inhibitors of mitochondrial complex I (NADH-ubiquinone oxidoreductase), are useful for elucidating the structural and functional features of the terminal electron transfer step of this enzyme. Previous studies of the structure-activity relationship revealed that except for the alkyl spacer linking the two toxophores (i.e., the hydroxylated THF and the gamma-lactone rings), none of the multiple functional groups of these inhibitors is essential for potent inhibition. To elucidate the function of the alkyl spacer, two sets of systematically selected analogues were synthesized. First, the length of the spacer was varied widely. Second, the local flexibility of the spacer was specifically reduced by introducing multiple bond(s) into different regions of the spacer. The optimal length of the spacer for inhibition was approximately 13 carbon atoms. The decrease in the strength of the inhibitory effect caused by elongating the spacer from 13 carbons was much more drastic than that caused by shortening. Local flexibility in a specific region of the spacer was not important for the inhibition. These observations indicate that the active conformation of the spacer is not an extended form, and is not necessarily restricted to a certain rigid shape. Moreover, an analogue in which a spacer covering 10 carbon atoms was hardened into a rodlike shape still maintained a potent inhibitory effect. Our results strongly suggest that the spacer portion is free from steric congestion arising from the putative binding site probably because there is no cavity-like binding site for the spacer portion. The manner of acetogenin binding to the enzyme may not be explained by a simple "key and keyhole" analogy.     

Cholestatic liver fibrosis and toxin-induced fibrosis are exacerbated in matrix metalloproteinase-2 deficient mice

Matrix metalloproteinase (MMP) plays an important role in homeostatic regulation of the extracellular environment and degradation of matrix. During liver fibrosis, several MMPs, including MMP-2, are up-regulated in activated hepatic stellate cells, which are responsible for exacerbation of liver cirrhosis. However, it remains unclear how loss of MMP-2 influences molecular dynamics associated with fibrogenesis in the liver. To explore the role of MMP-2 in hepatic fibrogenesis, we employed two fibrosis models in mice; toxin (carbon tetrachloride, CCl4)-induced and cholestasis-induced fibrosis. In the chronic CCl4 administration model, MMP-2 deficient mice exhibited extensive liver fibrosis as compared with wild-type mice. Several molecules related to activation of hepatic stellate cells were up-regulated in MMP-2 deficient liver, suggesting that myofibroblastic change of hepatic stellate cells was promoted in MMP-2 deficient liver. In the cholestasis model, fibrosis in MMP-2 deficient liver was also accelerated as compared with wild type liver. Production of tissue inhibitor of metalloproteinase 1 increased in MMP-2 deficient liver in both models, while transforming growth factor β, platelet-derived growth factor receptor and MMP-14 were up-regulated only in the CCl4 model. Our study demonstrated, using 2 experimental murine models, that loss of MMP-2 exacerbates liver fibrosis, and suggested that MMP-2 suppresses tissue inhibitor of metalloproteinase 1 up-regulation during liver fibrosis.     

Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to prevent the development of liver fibrosis in a number of pre-clinical studies. Marked changes in liver histopathology and serological markers of liver function have been observed without a clear understanding of the therapeutic mechanism by which stem cells act. We sought to determine if MSCs could modulate the activity of resident liver cells, specifically hepatic stellate cells (SCs) by paracrine mechanisms using indirect cocultures. Indirect coculture of MSCs and activated SCs led to a significant decrease in collagen deposition and proliferation, while inducing apoptosis of activated SCs. The molecular mechanisms underlying the modulation of SC activity by MSCs were examined. IL-6 secretion from activated SCs induced IL-10 secretion from MSCs, suggesting a dynamic response of MSCs to the SCs in the microenvironment. Blockade of MSC-derived IL-10 and TNF-alpha abolished the inhibitory effects of MSCs on SC proliferation and collagen synthesis. In addition, release of HGF by MSCs was responsible for the marked induction of apoptosis in SCs as determined by antibody-neutralization studies. These findings demonstrate that MSCs can modulate the function of activated SCs via paracrine mechanisms provide a plausible explanation for the protective role of MSCs in liver inflammation and fibrosis, which may also be relevant to other models of tissue fibrosis.     

Dictyostelium myosin II mutations that uncouple the converter swing and ATP hydrolysis cycle

During the ATP hydrolysis cycle of the Dictyostelium myosin II motor domain, two conserved alpha-helices, the SH1/SH2 helix and the relay helix, rotate in a coordinated way to induce the swing motion of the converter domain. A network of hydrophobic and ionic interactions in these two helices and the converter may ensure that the motions of these helices are effectively transmitted to the converter. To examine the roles of these interactions in the ATPase-dependent converter swing, we disrupted two conserved hydrophobic linkages among them by means of a point mutation (I499A or F692A). The resulting mutations induced only limited changes in the kinetic parameters of ATP hydrolysis, except for a marked increase of basal MgATPase activity. However, the mutant myosins completely lost their in vitro and in vivo motor functions. Measurements of the intrinsic tryptophan fluorescence and the GFP-based FRET revealed that the converter domain of these mutants did not swing during steady-state ATP hydrolysis or in the presence of tightly trapped Mg.ADP.V(i), which shows that the point mutations induced the uncoupling of the converter swing and ATP hydrolysis cycle. These results highlight the importance of these hydrophobic linkages for transmitting the coordinated twist motions of the helices to the converter as well as the requirement of this converter swing for force generation.     

Association of lecithin-cholesterol acyltransferase activity and low-density lipoprotein heterogeneity with atherosclerotic cardiovascular disease risk: a longitudinal pilot study

Background

Lecithin-cholesterol acyltransferase (LCAT) is believed to be involved in reverse cholesterol transport, which is known to play a key role in suppression of atherosclerosis. However, recent investigations have demonstrated that higher LCAT activity, measured in terms of the serum cholesterol esterification rate by an endogenous substrate method, is associated with increased formation of triglyceride (TG)-rich lipoproteins (TRLs), leading to a decrease in the low-density lipoprotein (LDL) particle size. The purpose of this hospital-based longitudinal study was to clarify the causal relationship between changes in the LCAT activity and changes in the LDL-particle size.

Methods

The subjects were a total of 335 patients, derived from our previous study cohort, with one or more risk factors for atherosclerotic cardiovascular disease (ASCVD). For this study, we measured the LDL-particle size (relative LDL migration [LDL-Rm value]) by polyacrylamide gel electrophoresis in the subjects, along with the changes in the LCAT activity, at the end of a follow-up period of at least 1 year.

Results

The results revealed that the absolute change (Δ) in the LDL-particle size increased significantly as the quartile of Δ LCAT activity increased (p =?0.01). A multi-logistic regression adjusted-analysis revealed that Δ LCAT activity in the fourth quartile as compared to that in the first quartile was independently predictive of an increased LDL-particle size (odds ratio [95% confidence interval]: 2.03 [1.02/4.04], p =?0.04). Moreover, the ? LCAT activity was also positively correlated with ? TRL-related markers (i.e., TG, remnant particle-like cholesterol [RLP-C], apolipoprotein B, apolipoprotein C-2, and apolipoprotein C-3).

Conclusions

The results lend support to the hypothesis that increased LCAT activity may be associated with increased formation of TRLs, leading to a reduction in the LDL-particle size in patients at a high risk for ASCVD. To reduce the risk of ASCVD, it may be important to focus not only on the quantitative changes in the serum LDL-cholesterol levels, but also on the LCAT activity.

Trial registration

UMIN (https://upload.umin.ac.jp/cgi-bin/ctr/ctr_reg_list.cgi) Study ID: UMIN000033228 retrospectively registered 2 July 2018.

     

Platelets Promote Tumor Growth and Metastasis via Direct Interaction between Aggrus/Podoplanin and CLEC-2

The platelet aggregation-inducing factor Aggrus, also known as podoplanin, is frequently upregulated in several types of tumors and enhances hematogenous metastasis by interacting with and activating the platelet receptor CLEC-2. Thus, Aggrus–CLEC-2 binding could be a therapeutic molecular mechanism for cancer therapy. We generated a new anti-human Aggrus monoclonal antibody, MS-1, that suppressed Aggrus–CLEC-2 binding, Aggrus-induced platelet aggregation, and Aggrus-mediated tumor metastasis. Interestingly, the MS-1 monoclonal antibody attenuated the growth of Aggrus-positive tumors in vivo. Moreover, the humanized chimeric MS-1 antibody, ChMS-1, also exhibited strong antitumor activity against Aggrus-positive lung squamous cell carcinoma xenografted into NOD-SCID mice compromising antibody-dependent cellular cytotoxic and complement-dependent cytotoxic activities. Because Aggrus knockdown suppressed platelet-induced proliferation in vitro and tumor growth of the lung squamous cell carcinoma in vivo, Aggrus may be involved in not only tumor metastasis but also tumor growth by promoting platelet-tumor interaction, platelet activation, and secretion of platelet-derived factors in vivo. Our results indicate that molecular target drugs inhibiting specific platelet–tumor interactions can be developed as antitumor drugs that suppress both metastasis and proliferation of tumors such as lung squamous cell carcinoma.