Interferon lambda 4 genotypes and resistance-associated variants in patients infected with hepatitis C virus genotypes 1 and 3.

UNLABELLED: Single-nucleotide polymorphisms (SNPs) in the interferon lambda 4 (IFNL4) gene are predictors for treatment success in patients with hepatitis C virus (HCV) infection. For direct-acting antiviral combinations only weak association with IFNL4 SNPs was observed. Little is known about potential selections of resistance-associated variants (RAVs) by the IFNL4 genotype. This study analyzed the prevalence of RAVs to currently approved direct-acting antivirals in a large European population in correlation to SNPs in IFNL4. Samples of 633 patients chronically infected with HCV genotypes 1a (n = 259), 1b (n = 323), and 3 (n = 51) were genotyped for rs12979860 (formerly known as IL28B) and rs368234815. RAVs in NS3, NS5A, and NS5B were detected by population-based sequencing. In addition, IFNL4 SNPs and NS5A RAVs were analyzed including deep sequencing (n = 109) in an independent replication cohort of HCV genotype 1-infected patients (n = 201). No significant correlation was found between IFNL4 SNPs and rare and common RAVs within NS3 and NS5B. In contrast, the NS5A RAV Y93H was detected frequently in HCV genotype 1b (14%) and significantly associated with the beneficial IFNL4 SNPs (P < 0.001 and P = 0.002, respectively). Moreover, the presence of Y93H in HCV genotype 1b patients was significantly associated with the second site variant T83M (P < 0.001). Independent factors significantly associated with the presence of Y93H were IFNL4 genotype and high baseline viral load. CONCLUSION: The NS5A RAV Y93H is significantly associated with the presence of beneficial IFNL4 SNPs and a high baseline viral load in HCV genotype 1-infected patients, which may explain a lack of correlation or even an inverse correlation of treatment response with IFNL4 genotype in some NS5A inhibitor containing IFN-free regimens.

Capillary pCO2 helps distinguishing idiopathic pulmonary arterial hypertension from pulmonary hypertension due to heart failure with preserved ejection fraction.

RATIONALE: The demographics of patients with idiopathic pulmonary arterial hypertension (IPAH) are changing and this diagnosis is increasingly being made in older patients. However, diagnostic misclassifications are common as it may be difficult to differentiate between IPAH and pulmonary hypertension due to heart failure with preserved ejection fraction (PH-HFpEF). We investigated the hypothesis that the capillary pCO2 (pcCO2) may help distinguishing between idiopathic pulmonary arterial hypertension (IPAH) and pulmonary hypertension due to heart failure with preserved ejection fraction (PH-HFpEF). METHODS: In a cross-sectional study, we retrospectively assessed pcCO2 levels (obtained from arterialized capillary blood at the time of diagnosis) from patients with IPAH or PH-HFpEF, respectively. Receiver operated characteristics (ROC) were used to determine the pcCO2 level providing the best discrimination between these two conditions. PcCO2 values were considered helpful if they were associated with a negative predictive value >0.9 to excluded either IPAH or PH-HFpEF. RESULTS: The study enrolled 185 patients, 99 with IPAH (74% female; age 47 ± 17 years; body mass index 26 ± 5 kg/m2, PAPm 53 ± 12 mmHg, PAWP 8 ± 3 mmHg), and 86 with PH-HFpEF (64% female; age 69 ± 10 years; body mass index 30 ± 6 kg/m2, PAPm 47 ± 10 mmHg, PAWP 21 ± 5 mmHg). PcCO2 at time of diagnosis was 33 ± 4 mmHg in the IPAH group and 40 ± 5 mmHg in the PH-HFpEF group (p < 0.001), respectively. According to ROC analysis, a pcCO2 of 36 mmHg was the best discriminator between both entities with an area under curve of 0.87 (p < 0.001). The likelihood of PH-HFpEF was <10% in patients with a PcCO2 < 34 mmHg, whereas the likelihood of IPAH was <10% in patients with a PcCO2 > 41 mmHg. CONCLUSIONS: PcCO2 levels were significantly lower in IPAH compared to PH-HFpEF and may provide useful information in differentiating between both conditions.

NMR- and circular dichroism-monitored lipid binding studies suggest a general role for the FATC domain as membrane anchor of phosphatidylinositol 3-kinase-related kinases (PIKK).

The FATC domain is shared by all members of the family of phosphatidylinositol-3 kinase-related kinases (PIKKs). It has been shown that the FATC domain plays an important role for the regulation of each PIKK. However, other than an involvement in protein-protein interactions, a common principle for the action of the FATC domain has not been detected. A detailed characterization of the structure and lipid binding properties of the FATC domain of the Ser/Thr kinase target of rapamycin (TOR) revealed that it contains a redox-sensitive membrane anchor in its C terminus. Because the C-terminal regions of the FATC domains of all known PIKKs are rather hydrophobic and especially rich in aromatic residues, we examined whether the ability to interact with lipids and membranes might be a general property. Here, we present the characterization of the interactions with lipids and different membrane mimetics for the FATC domains of human DNA-PKcs, human ATM, human ATR, human SMG-1, and human TRRAP by NMR and CD spectroscopy. The data indicate that all of these can interact with different membrane mimetics and may have different preferences only for membrane properties such as surface charge, curvature, and lipid packing. The oxidized form of the TOR FATC domain is well structured overall and forms an α-helix that is followed by a disulfide-bonded loop. In contrast, the FATC domains of the other PIKKs are rather unstructured in the isolated form and only significantly populate α-helical secondary structure upon interaction with membrane mimetics.

1H, 15N, and 13C chemical shift assignments of the micelle immersed FAT C-terminal (FATC) domains of the human protein kinases ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) fused to the B1 domain of streptococcal protein G (GB1).

FAT C-terminal (FATC) is a circa 33 residue-long domain. It controls the kinase functionality in phosphatidylinositol-3 kinase-related kinases (PIKKs). Recent NMR- and CD-monitored interaction studies indicated that the FATC domains of all PIKKs can interact with membrane mimetics albeit with different preferences for membrane properties such as surface charge and curvature. Thus they may generally act as membrane anchoring unit. Here, we present the 1H, 15N, and 13C chemical shift assignments of the DPC micelle immersed FATC domains of the human PIKKs ataxia-telangiectasia mutated (ATM, residues 3024-3056) and DNA protein kinase catalytic subunit (DNA-PKcs, residues 4096-4128), both fused to the 56 residue long B1 domain of Streptococcal protein G (GB1). Each fusion protein is 100 amino acids long and contains in the linking region between the GB1 tag and the FATC region a thrombin (LVPRGS) and an enterokinase (DDDDK) protease site. The assignments pave the route for the detailed structural characterization of the membrane mimetic bound states, which will help to better understand the role of the proper cellular localization at membranes for the function and regulation of PIKKs. The chemical shift assignment of the GB1 tag is useful for NMR spectroscopists developing new experiments or using GB1 otherwise for case studies in the field of in-cell NMR spectroscopy or protein folding. Moreover it is often used as purification tag. Earlier we showed already that GB1 does not interact with membrane mimetics and thus does not disturb the NMR monitoring of membrane mimetic interactions of attached proteins.

Comparative characterization of hepatitis B virus surface antigen derived from different hepatitis B virus genotypes.

For human hepatitis B virus eight distinct and two candidate genotypes are described. These genotypes differ with respect to geographic distribution, molecular virology and virus-associated pathogenesis. Comparative analysis of HBV genotypes revealed, with exception of HBV/G that shows impaired HBsAg release, that no fundamental disparities between genotypes exist regarding glycosylation, subcellular distribution, release of HBsAg and formation of subviral particles. However, there are distinctions regarding the proportion of L to M to S HBs proteins detected intra- and extracellularly for different genotypes. 2D electrophoresis revealed different posttranslational modification patterns for LHBs. In light of the relevance of HBsAg as diagnostic marker, detectability of purified recombinant HBsAg of various genotypes by HBsAg-specific detection systems licensed in Europe was investigated, showing similar sensitivities for genotypes included in this analysis. These data indicate that recombinant HBsAg reproducibly purified following a defined protocol might be used as an alternative to reference materials currently established.

Characterization of residue-dependent differences in the peripheral membrane association of the FATC domain of the kinase 'target of rapamycin' by NMR and CD spectroscopy.

The conserved C-terminal FATC domain of the kinase 'target of rapamycin' is important for its regulation and was suggested to contain a peripheral membrane anchor. Here, we present the characterization of the interactions of the yeast TOR1 FATC domain (2438-2470=y1fatc) and 15 mutants with membrane mimetic micelles, bicelles, and small unilamellar vesicles (SUVs) by NMR and CD spectroscopy. Replacement of up to 6-7 residues did not result in a significant abrogation of the association with micelles or bicelles. However, replacement of only one residue could result in an impairment of the interaction with SUVs that are usually used at low concentrations. Some mutants not binding liposomes may be introduced in full-length TOR for future functional and localization studies in vivo.

Characterization of the immersion properties of the peripheral membrane anchor of the FATC domain of the kinase "target of rapamycin" by NMR, oriented CD spectroscopy, and MD simulations.

The multidomain ser/thr kinase "target of rapamycin" (TOR) centrally controls eukaryotic growth and metabolism. The C-terminal FATC domain is important for TOR regulation and was suggested to directly mediate TOR-membrane interactions. Here, we present a detailed characterization of the membrane immersion properties of the oxidized and reduced yeast TOR1 FATC domain (2438-2470 = y1fatc). The immersion depth was characterized by NMR-monitored interaction studies with DPC micelles containing paramagnetically tagged 5- or 16-doxyl stearic acid (5-/16-SASL) and by analyzing the paramagnetic relaxation enhancement (PRE) from Mn(2+) in the solvent. Complementary MD-simulations of micellar systems in the absence and presence of protein showed that 5-/16-SASL can move in the micelle and that 16-SASL can bend such that the doxyl group is close to the headgroup region and not deep in the interior as commonly assumed. Based on oriented CD (OCD) data, the single α-helix of oxidized/reduced y1fatc has an angle to the membrane normal of ∼30-60°/∼35-65° in neutral and ∼5-35°/∼0-30° in negatively charged bilayers. The presented experimentally well-founded models help to better understand how this redox-sensitive peripheral membrane anchor may be part of a network of protein-protein and protein-membrane interactions regulating TOR localization at different cellular membranes. Moreover, the presented work provides a good methodological reference for the structural characterization of other peripherally membrane associating proteins.

A fast and simple method for probing the interaction of peptides and proteins with lipids and membrane-mimetics using GB1 fusion proteins and NMR spectroscopy.

The expression of peptides and proteins as fusions to the B1 domain of streptococcal protein G (GB1) is very popular since GB1 often improves the solubility of the target protein and because the first purification step using IgG affinity chromatography is simple and efficient. However, the following protease digest is not always complete or can result in a digest of the target protein. In addition, a further purification step such as RP-HPLC has to be used to get rid of the GB1 tag and undigested fusion protein. Because the protease digest and the following purification step are not only time-consuming but generally also expensive, we tested if GB1 fusion proteins can directly be used for NMR interaction studies using lipids or membrane-mimetics. Based on NMR binding studies using only the GB1 part, this fusion tag does not significantly interact with different membrane-mimetics such as micelles, bicelles, or liposomes. Thus spectral changes observed using GB1-fusion proteins indicate lipid- and membrane interactions of the target protein. The method was initially established to probe membrane interactions of a large number of mutants of the FATC domain of the ser/thr kinase TOR. To demonstrate the usefulness of the approach, we show NMR binding data for the wild type protein and a leucine to alanine mutant.