Aberrant splicing in MLH1 and MSH2 due to exonic and intronic variants

Single base substitutions in DNA mismatch repair genes which are predicted to lead either to missense or silent mutations, or to intronic variants outside the highly conserved splicing region are often found in hereditary nonpolyposis colorectal cancer (HNPCC) families. In order to use the variants for predictive testing in persons at risk, their pathogenicity has to be evaluated. There is growing evidence that some substitutions have a detrimental influence on splicing. We examined 19 unclassified variants (UVs) detected in MSH2 or MLH1 genes in patients suspected of HNPCC for expression at RNA level. We demonstrate that 10 of the 19 UVs analyzed affect splicing. For example, the substitution MLH1,c.2103G>C in the last position of exon 18 does not result in a missense mutation as theoretically predicted (p.Gln701His), but leads to a complete loss of exon 18. The substitution MLH1,c.1038G>C (predicted effect p.Gln346His) leads to complete inactivation of the mutant allele by skipping of exons 10 and 11, and by activation of a cryptic intronic splice site. Similarly, the intronic variant MLH1,c.306+2dupT results in loss of exon 3 and a frameshift mutation due to a new splice donor site 5 bp upstream. Furthermore, we confirmed complete exon skipping for the mutations MLH1,c.1731G>A and MLH1,c.677G>A. Partial exon skipping was demonstrated for the mutations MSH2,c.1275A>G, MLH1,c.588+5G>A, MLH1,c.790+4A>G and MLH1,c.1984A>C. In contrast, five missense mutations (MSH2,c.4G>A, MSH2,c.2123T>A, MLH1,c.464T>G, MLH1,c.875T>C and MLH1,c.2210A>T) were found in similar proportions in the mRNA as in the genomic DNA. We conclude that the mRNA examination should precede functional tests at protein level. Databases: HNPCC – OMIM 114500, MSH2 – OMIM: 120435; GenBank: NM_000251.1, MLH1 – OMIM: 120436; GenBank: NM_000249.2, InSiGHT mutation database: , Programs: BDGP: , ESEfinder program:     

Myeloperoxidase impairs ABCA1-dependent cholesterol efflux through methionine oxidation and site-specific tyrosine chlorination of apolipoprotein A-I

High density lipoprotein (HDL) isolated from human atherosclerotic lesions and the blood of patients with established coronary artery disease contains elevated levels of 3-chlorotyrosine. Myeloperoxidase (MPO) is the only known source of 3-chlorotyrosine in vivo, indicating that MPO oxidizes HDL in humans. We previously reported that Tyr-192 is the major site that is chlorinated in apolipoprotein A-I (apoA-I), the chief protein in HDL, and that chlorinated apoA-I loses its ability to promote cholesterol efflux from cells by the ATP-binding cassette transporter A1 (ABCA1) pathway. However, the pathways that promote the chlorination of specific Tyr residues in apoA-I are controversial, and the mechanism for MPO-mediated loss of ABCA1-dependent cholesterol efflux of apoA-I is unclear. Using site-directed mutagenesis, we now demonstrate that lysine residues direct tyrosine chlorination in apoA-I. Importantly, methionine residues inhibit chlorination, indicating that they can act as local, protein-bound antioxidants. Moreover, we observed near normal cholesterol efflux activity when Tyr-192 of apoA-I was mutated to Phe and the oxidized protein was incubated with methionine sulfoxide reductase. Thus, a combination of Tyr-192 chlorination and methionine oxidation is necessary for depriving apoA-I of its ABCA1-dependent cholesterol transport activity. Our observations suggest that biologically significant oxidative damage of apoA-I involves modification of a limited number of specific amino acids, raising the feasibility of producing oxidation-resistant forms of apoA-I that have enhanced anti-atherogenic activity in vivo.     

Platelets promote coagulation factor XII-mediated proteolytic cascade systems in plasma

Blood coagulation factor XII (FXII, Hageman factor) is a plasma serine protease which is autoactivated following contact with negatively charged surfaces in a reaction involving plasma kallikrein and high-molecular-weight kininogen (contact phase activation). Active FXII has the ability to initiate blood clotting via the intrinsic pathway of coagulation and inflammatory reactions via the kallikrein-kinin system. Here we have determined FXII-mediated bradykinin formation and clotting in plasma. Western blotting analysis with specific antibodies against various parts of the contact factors revealed that limited activation of FXII is sufficient to promote plasma kallikrein activation, resulting in the conversion of high-molecular-weight kininogen and bradykinin generation. The presence of platelets significantly promoted FXII-initiated bradykinin formation. Similarly, in vitro clotting assays revealed that platelets critically promoted FXII-driven thrombin and fibrin formation. In summary, our data suggest that FXII-initiated protease cascades may proceed on platelet surfaces, with implications for inflammation and clotting.     

Defects in N-glycosylation induce apoptosis in yeast

N-glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to man. Defects of N-glycosylation in humans lead to congenital disorders. The pivotal step of this pathway is the transfer of the evolutionarily conserved lipid-linked core-oligosaccharide to the nascent polypeptide chain, catalysed by the oligosaccharyltransferase. One of its nine subunits, Ost2, has homology to DAD1, originally characterized in hamster cells as a defender against apoptotic death. Here we show that ost mutants, such as ost2 and wbp1-1, display morphological and biochemical features of apoptosis upon induction of the glycosylation defect. We observe nuclear condensation, DNA fragmentation as well as externalization of phosphatidylserine. We also demonstrate induction of caspase-like activity, both determined by flow cytometric analysis and in cell-free extracts. Similarly, the N-glycosylation inhibitor tunicamycin in combination with elevated temperature is able to challenge the apoptotic cascade. Heterologous expression of anti-apoptotic human Bcl-2 diminishes caspase activation, improves survival of cells and suppresses the temperature-sensitive growth defect of wbp1-1. Furthermore, accumulation of reactive oxygen species occurs in response to defective glycosylation. As deletion of the metacaspase YCA1 does not seem to abrogate glycosylation-induced apoptosis, we postulate a different proteolytic process to be involved in this death pathway.     

Lithium induces clearance of protease resistant prion protein in prion-infected cells by induction of autophagy

Lithium is used for several decades to treat manic-depressive illness (bipolar affective disorder). Recently, it was found that lithium induces autophagy, thereby promoting the clearance of mutant huntingtin and α-synucleins in experimental systems. We show here for the first time that lithium significantly reduces the amount of pathological prion protein (PrP^Sc) in prion-infected neuronal and non-neuronal cultured cells by inducing autophagy. Treatment of prion-infected cells with 3-methyladenine, a potent inhibitor of autophagy, counteracted the anti-prion effect of lithium, demonstrating that induction of autophagy mediates degradation of PrP^Sc. Co-treatment with lithium and rapamycin, a drug widely used to induce autophagy, had an additive effect on PrP^Sc clearance compared to treatment with either drug alone. In addition, we provide evidence that the ability to reduce PrP^Sc and to induce autophagy is common for diverse lithium compounds, not only for the drug lithium chloride, usually administered in clinical therapy. Furthermore, we show here that besides reduction of PrP^Sc-aggregates, lithium-induced autophagy also slightly reduces the levels of cellular prion protein. Limiting the substrate available for conversion of cellular prion protein into PrP^Sc may provide an additional mechanism for reduction of PrP^Sc by lithium-induced autophagy.     

A-type carrier protein ErpA is essential for formation of an active formate-nitrate respiratory pathway in Escherichia coli K-12

A-type carrier (ATC) proteins of the Isc (iron-sulfur cluster) and Suf (sulfur mobilization) iron-sulfur ([Fe-S]) cluster biogenesis pathways are proposed to traffic preformed [Fe-S] clusters to apoprotein targets. In this study, we analyzed the roles of the ATC proteins ErpA, IscA, and SufA in the maturation of the nitrate-inducible, multisubunit anaerobic respiratory enzymes formate dehydrogenase N (Fdh-N) and nitrate reductase (Nar). Mutants lacking SufA had enhanced activities of both enzymes. While both Fdh-N and Nar activities were strongly reduced in an iscA mutant, both enzymes were inactive in an erpA mutant and in a mutant unable to synthesize the [Fe-S] cluster scaffold protein IscU. It could be shown for both Fdh-N and Nar that loss of enzyme activity correlated with absence of the [Fe-S] cluster-containing small subunit. Moreover, a slowly migrating form of the catalytic subunit FdnG of Fdh-N was observed, consistent with impeded twin arginine translocation (TAT)-dependent transport. The highly related Fdh-O enzyme was also inactive in the erpA mutant. Although the Nar enzyme has its catalytic subunit NarG localized in the cytoplasm, it also exhibited aberrant migration in an erpA iscA mutant, suggesting that these modular enzymes lack catalytic integrity due to impaired cofactor biosynthesis. Cross-complementation experiments demonstrated that multicopy IscA could partially compensate for lack of ErpA with respect to Fdh-N activity but not Nar activity. These findings suggest that ErpA and IscA have overlapping roles in assembly of these anaerobic respiratory enzymes but demonstrate that ErpA is essential for the production of active enzymes.     

Prevailing sea surface temperatures inhibit summer reproduction of the kelp Laminaria digitata at Helgoland (North Sea)

The impact of abiotic factors on kelp sporophyte reproduction has rarely been investigated. Laminaria digitata (Hudson) J.V. Lamouroux is one of the few summer fertile Laminaria species worldwide and reproduction is subjected to relatively high water temperatures. We investigated the impact of prevailing summer temperatures (~18°C in August) on the induction of sporangia, meiospore release, and germination at the island of Helgoland (North Sea). At Helgoland, fertile sporophytes are found between April and December with a maximum in late summer. While released meiospore numbers were constant between June and October, germination rates decreased significantly in summer. Short‐term exposure of mature sori to 17°C–22°C induced a significantly higher meiospore release indicating enhancement of sporulation by elevated temperatures. Induction of sporangia on vegetative blade disks was not possible at 20°C, and fertility was only 20% at 18°C–19°C, but it was 100% in cool temperatures of 1°C–10°C. It was shown for the first time in a kelp species that “sporogenesis” is the life‐cycle process with the narrowest temperature window compared to growth or survival of the sporophyte or reproduction, growth, and survival of the gametophyte. We incorporated several parameters (induction time, fertile area, and relative fertility) into a “Reproductive efficiency index.” This indicates that sporogenesis of L. digitata is a cold‐adapted process with an optimum at (5)–10°C. The results show that the population at Helgoland is at its reproduction limit despite the existence of other geographically more southerly located populations.     

Validation of an isotope dilution gas chromatography-mass spectrometry method for analysis of 7-oxygenated campesterol and sitosterol in human serum

High dose daily intake of plant sterols decreases the uptake of cholesterol in the intestine by competitive mechanisms and thus leads to reduced serum levels of total and LDL-cholesterol. By this, the commercialization of plant sterol enriched ‘functional food’ products is rapidly increasing. Subjects using these kinds of diet present a duplication of their serum plant sterol levels after long-term intake. In analogy to cholesterol, plant sterols such as campesterol and sitosterol can be oxidized to oxyphytosterols and these may counteract the primary anti-atherosclerotic action of cholesterol lowering. In order to investigate the whole spectrum of the consequences following high plant sterol intake a highly sensitive and specific isotope dilution gas chromatography–mass spectrometry method for the analysis of 7-oxygenated campesterol/sitosterol in trace amounts in human serum is presented in this paper. The validation was based on limits for detection and quantification, recovery, precision and minimization of autoxidation during work-up. Our results show an overall coefficient of variation ≤10% for the precision. The lowest limits for detection and quantification for 7α-hydroxy-campesterol were 7 pg/mL and 23 pg/mL, respectively. Data for overall sum recovery ranged from 92% to 115%. We practically used this method for analysis of oxyphytosterols simultaneously with plant sterol concentrations in serum from healthy volunteers. Sixteen subjects were treated with plant sterol enriched margarine (3 g/day) for 28 days. The results showed a significant increase of the oxyphytosterol 7β-hydroxy-sitosterol from 1.19 ± 0.54 (before intake) to 2.24 ± 1.24 ng/mL (mean ± SD; +86.7%; P = 0.007) after intake of the margarine. There was a highly significant correlation between the serum levels of campesterol and the sum of 7-oxygenated campesterol (R² = 0.915; P < 0.001) and sitosterol and the sum of 7-oxygenated sitosterol (R² = 0.915; P < 0.001). We can conclude from this study that the analytic method is well suited for detection of OPS, even at trace amounts.     

Dependence on the F<Subscript>0</Subscript>F<Subscript>1</Subscript>-ATP synthase for the activities of the hydrogen-oxidizing hydrogenases 1 and 2 during glucose and glycerol fermentation at high and low pH in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli has four [NiFe]-hydrogenases (Hyd); three of these, Hyd-1, Hyd-2 and Hyd-3 have been characterized well. In this study the requirement for the F₀F₁-ATP synthase for the activities of the hydrogen-oxidizing hydrogenases Hyd-1 and Hyd-2 was examined. During fermentative growth on glucose at pH 7.5 an E. coli F₀F₁-ATP synthase mutant (DK8) lacked hydrogenase activity. At pH 5.5 hydrogenase activity was only 20% that of the wild type. Using in-gel activity staining, it could be demonstrated that both Hyd-1 and Hyd-2 were essentially inactive at these pHs, indicating that the residual activity at pH 5.5 was due to the hydrogen-evolving Hyd-3 enzyme. During fermentative growth in the presence of glycerol, hydrogenase activity in the mutant was highest at pH 7.5 attaining a value of 0.76 U/mg, or ~50% of wild type activity, and Hyd-2 was only partially active at this pH, while Hyd-1 was inactive. Essentially no hydrogenase activity was measured at pH 5.5 during growth with glycerol. At this pH the mutant had a hydrogenase activity that was maximally only ~10% of wild type activity with either carbon substrate but a weak activity of both Hyd-1 and Hyd-2 could be detected. Taken together, these results demonstrate for the first time that the activity of the hydrogen-oxidizing hydrogenases in E. coli depends on an active F₀F₁-ATP synthase during growth at high and low pH.     

NompC TRP channel is essential for Drosophila sound receptor function

The idea that the NompC TRPN1 channel is the Drosophila transducer for hearing has been challenged by remnant sound-evoked nerve potentials in nompC nulls. We now report that NompC is essential for the function of Drosophila sound receptors and that the remnant nerve potentials of nompC mutants are contributed by gravity/wind receptor cells. Ablating the sound receptors reduces the amplitude and sensitivity of sound-evoked nerve responses, and the same effects ensued from mutations in nompC. Ablating the sound receptors also suffices to abolish mechanical amplification, which arises from active receptor motility, is linked to transduction, and also requires NompC. Calcium imaging shows that the remnant nerve potentials in nompC mutants are associated with the activity of gravity/wind receptors and that the sound receptors of the mutants fail to respond to sound. Hence, Drosophila sound receptors require NompC for mechanical signal detection and amplification, demonstrating the importance of this transient receptor potential channel for hearing and reviving the idea that the fly's auditory transducer might be NompC.