Important functional role of residue x of the presenilin GxGD protease active site motif for APP substrate cleavage specificity and substrate selectivity of γ‐secretase

γ‐Secretase plays a central role in the generation of the Alzheimer disease‐causing amyloid β‐peptide (Aβ) from the β‐amyloid precursor protein (APP) and is thus a major Alzheimer′s disease drug target. As several other γ‐secretase substrates including Notch1 and CD44 have crucial signaling functions, an understanding of the mechanism of substrate recognition and cleavage is key for the development of APP selective γ‐secretase‐targeting drugs. The γ‐secretase active site domain in its catalytic subunit presenilin (PS) 1 has been implicated in substrate recognition/docking and cleavage. Highly critical in this process is its GxGD active site motif, whose invariant glycine residues cannot be replaced without causing severe functional losses in substrate selection and/or cleavage efficiency. Here, we have investigated the contribution of the less well characterized residue x of the motif (L383 in PS1) to this function. Extensive mutational analysis showed that processing of APP was overall well‐tolerated over a wide range of hydrophobic and hydrophilic mutations. Interestingly, however, most L383 mutants gave rise to reduced levels of Aβ_37–39 species, and several increased the pathogenic Aβ_42/43 species. Several of the Aβ_42/43‐increasing mutants severely impaired the cleavages of Notch1 and CD44 substrates, which were not affected by any other L383 mutation. Our data thus establish an important, but compared with the glycine residues of the motif, overall less critical functional role for L383. We suggest that L383 and the flanking glycine residues form a spatial arrangement in PS1 that is critical for docking and/or cleavage of different γ‐secretase substrates.     

Substrate Binding Stabilizes a Pre-translocation Intermediate in the ATP-binding Cassette Transport Protein MsbA

ATP-binding cassette (ABC) transporters belong to one of the largest protein superfamilies that expands from prokaryotes to man. Recent x-ray crystal structures of bacterial and mammalian ABC exporters suggest a common alternating access mechanism of substrate transport, which has also been biochemically substantiated. However, the current model does not yet explain the coupling between substrate binding and ATP hydrolysis that underlies ATP-dependent substrate transport. In our studies on the homodimeric multidrug/lipid A ABC exporter MsbA from Escherichia coli, we performed cysteine cross-linking, fluorescence energy transfer, and cysteine accessibility studies on two reporter positions, near the nucleotide-binding domains and in the membrane domains, for transporter embedded in a biological membrane. Our results suggest for the first time that substrate binding by MsbA stimulates the maximum rate of ATP hydrolysis by facilitating the dimerization of nucleotide-binding domains in a state, which is markedly distinct from the previously described nucleotide-free, inward-facing and nucleotide-bound, outward-facing conformations of ABC exporters and which binds ATP.     

Secretory production of an FAD cofactor-containing cytosolic enzyme (sorbitol–xylitol oxidase from Streptomyces coelicolor) using the twin-arginine translocation (Tat) pathway of Corynebacterium glutamicum

Carbohydrate oxidases are biotechnologically interesting enzymes that require a tightly or covalently bound cofactor for activity. Using the industrial workhorse Corynebacterium glutamicum as the expression host, successful secretion of a normally cytosolic FAD cofactor-containing sorbitol–xylitol oxidase from Streptomyces coelicolor was achieved by using the twin-arginine translocation (Tat) protein export machinery for protein translocation across the cytoplasmic membrane. Our results demonstrate for the first time that, also for cofactor-containing proteins, a secretory production strategy is a feasible and promising alternative to conventional intracellular expression strategies.The secretory expression of recombinant proteins can offer significant process advantages over cytosolic production strategies, since secretion into the growth medium greatly facilitates downstream processing and therefore can significantly reduce the costs of producing a desired target protein (Quax, 1997). And, in fact, the enormous secretion capacity of certain Gram-positive bacteria (e.g. various Bacillus species) has been used since many years in industry for the production of mainly host-derived secretory proteins such as proteases and amylases, resulting in amounts of more than 20 g l⁻¹ culture medium (Harwood and Cranenburg, 2008). In contrast, attempts to use Bacillus species for the secretory production of heterologous proteins have often failed or led to disappointing results, a fact that, among other reasons, could in many cases be attributed to the presence of multiple cell wall-associated and secreted proteases that rapidly degraded the heterologous target proteins (Li et al., 2004; Sarvas et al., 2004; Westers et al., 2011). Therefore, an increasing need exists to explore alternative host systems with respect to their ability to express and secrete problematic and/or complex heterologous proteins of biotechnological interest.So far, the Gram-positive bacterium Corynebacterium glutamicum has been used in industry mainly for the production of amino acids and other low-molecular weight compounds (Leuchtenberger et al., 2005; Becker and Wittmann, 2011; Litsanov et al., 2012). However, various recent reports have indicated that C. glutamicum might likewise possess a great potential as an alternative host system for the secretory expression of foreign proteins. Corynebacterium glutamicum belongs to a class of diderm Gram-positive bacteria that, besides the cytoplasmic membrane, possess an additional mycolic acid-containing outer membrane-like structure that acts as an extremely efficient permeability barrier for hydrophilic compounds (Hoffmann et al., 2008; Zuber et al., 2008). Despite this fact, an efficient secretion of various heterologous proteins into the growth medium of this microorganism has been observed (e.g. Billman-Jacobe et al., 1995; Meissner et al., 2007; Kikuchi et al., 2009; Tateno et al., 2009; Tsuchidate et al., 2011).In bacteria, two major export pathways exist for the transport of proteins across the cytoplasmic membrane that fundamentally differ with respect to the folding status of their respective substrate proteins during the actual translocation step. The general secretion (Sec) system transports its substrates in a more or less unfolded state and folding takes places on the trans side of the membrane after the actual transport event (Yuan et al., 2010; du Plessis et al., 2011). In contrast, the alternative twin-arginine translocation (Tat) system translocates its substrates in a fully folded form and therefore provides an attractive alternative for the secretory production of proteins that cannot be produced in a functional form via the Sec route (Brüser, 2007). Carbohydrate oxidases are biotechnologically interesting enzymes (van Hellemond et al., 2006) that are excluded from Sec-dependent secretion since they depend on a tightly or covalently bound cofactor for their activity and, for this reason, require that their folding and cofactor insertion has to take place in the cytosol. Because C. glutamicum has shown to be an excellent host for the Tat-dependent secretion of the cofactor-less model protein GFP (Meissner et al., 2007; Teramoto et al., 2011), we now asked whether it is likewise possible to secrete a cofactor-containing enzyme into the supernatant of C. glutamicum using the same protein export route.As a model protein, we chose the sorbitol–xylitol oxidase (SoXy) from Streptomyces coelicolor, a normally cytosolic enzyme that possesses a covalently bound FAD molecule as cofactor (Heuts et al., 2007; Forneris et al., 2008). FAD is incorporated into the apoprotein in a post-translational and self-catalytic process that only occurs if the polypeptide chain has adopted a correctly folded structure (Heuts et al., 2007; 2009). To direct SoXy into the Tat export pathway of C. glutamicum, we constructed a gene encoding a TorA–SoXy hybrid precursor in which SoXy is fused to the strictly Tat-specific signal peptide of the periplasmic Escherichia coli Tat substrate trimethylamine N-oxide reductase (TorA) (Fig. 1) which, in our previous study, has been proven to be a functional and strictly Tat-specific signal peptide also in C. glutamicum (Meissner et al., 2007). The corresponding torA–soxy gene was cloned into the expression vector pEKEx2 (Eikmanns et al., 1991) under the control of an IPTG-inducible P_tac promotor. After transformation of the resulting plasmid pTorA–SoXy into the C. glutamicum ATCC13032 wild-type strain, two independent colonies of the resulting recombinant C. glutamicum (pTorA–SoXy) strain and, as a control, a colony of a strain that contained the empty expression vector without insert [C. glutamicum (pEKEx2)] were grown in CGXII medium (Keilhauer et al., 1993) at 30°C for 16 h in the presence of 1 mM IPTG. Subsequently, the proteins present in the culture supernatants were analysed by SDS-PAGE followed by staining with Coomassie blue. As shown in Fig. 2, in the supernatants of the pTorA–SoXy-containing cells (lanes 3 and 4), a prominent protein band of approximately 44 kDa can be detected, the size of which is very similar to the calculated molecular mass (44.4 kDa) of SoXy. Since this band is completely lacking in the supernatant of the control strain (lane 2), this strongly suggests that this band corresponds to SoXy that has been secreted into the culture supernatant of C. glutamicum (pTorA–SoXy). And, in fact, this suggestion was subsequently confirmed in a direct way by MALDI-TOF mass spectrometry after extraction of the protein out of the gel followed by tryptic digestion (Schaffer et al., 2001) (data not shown).Open in a separate windowFigure 1The TorA–SoXy hybrid precursor protein. Upper part: Schematic drawing of the relevant part of the pTorA–SoXy expression vector. P_tac, IPTG-inducible tac promotor. RBS, ribosome binding site. To maintain the authentic TorA signal peptidase cleavage site, the first four amino acids of the mature TorA protein (black bar) were retained in the TorA–SoXy fusion protein. White bar: TorA signal peptide (TorA^SP); grey bar: SoXy (amino acids 2–418). Lower part: Amino acid sequence of the signal peptide and early mature region of the TorA–SoXy hybrid precursor. The twin-arginine consensus motif of the TorA signal peptide is underlined. The four amino acids derived from mature TorA are shown in italics. The signal peptidase cleavage site is indicated by an arrowhead.Open in a separate windowFigure 2Secretion of SoXy into the growth medium of C. glutamicum. Cells of C. glutamicum ATCC13032 containing the empty vector pEKEx2 and two independently transformed colonies of C. glutamicum (pTorA–SoXy) were grown overnight in 5 ml of BHI medium (Difco) at 30°C. The cells were washed once with CGXII medium (Keilhauer et al., 1993) and inoculated to an OD₆₀₀ of 0.5 in 5 ml of fresh CGXII medium containing 1 mM IPTG. After 16 h of further growth at 30°C, the supernatant fractions were prepared as described previously (Meissner et al., 2007). Samples corresponding to an equal number of cells were subjected to SDS-PAGE followed by staining with Coomassie blue. Lane 1, molecular mass marker (kDa). Lane 2, C. glutamicum (pEKEx2); lanes 3 and 4, C. glutamicum (pTorA–SoXy). The position of the secreted SoXy protein is indicated by an arrow.Next, the supernatant of C. glutamicum (pTorA–SoXy) was analysed for SoXy enzyme activity by measuring the production of H₂O₂ that is formed during the enzymatic conversion of sorbitol to fructose (Meiattini, 1983). Six hours after induction of gene expression by 1 mM IPTG, an enzymatic activity of 10.3 ± 1.6 nmol min⁻¹ ml⁻¹ could be determined in the supernatant of C. glutamicum (pTorA–SoXy). In contrast, no such activity was found in the supernatant of the control strain C. glutamicum (pEKEx2). From these results we conclude that we have succeeded in the secretion of enzymatically active and therefore FAD cofactor-containing SoXy into the culture supernatant of C. glutamicum.Finally, we examined whether the secretion of SoXy had in fact occurred via the Tat pathway of C. glutamicum. Plasmid pTorA–SoXy was used to transform C. glutamcium ATCC13032 wild type and a C. glutamicum ΔTatAC mutant strain that lacks two essential components of the Tat transport machinery and therefore does not possess a functional Tat translocase (Meissner et al., 2007). The corresponding cells were grown in BHI medium (Difco) at 30°C in the presence of 1 mM IPTG for 6 h. Subsequently, the proteins present in the cellular and the supernatant fractions of the corresponding cells were analysed by SDS-PAGE followed by Western blotting using SoXy-specific antibodies. As shown in Fig. 3, polypeptides corresponding to the unprocessed TorA–SoXy precursor and some minor smaller degradation products of it can be detected in the cellular fractions of both the wild-type and the ΔTatAC deletion strains (lanes 3 and 5). In the supernatant fraction of the Tat⁺ wild-type strain (lane 4), but not that of the ΔTatAC strain (lane 6), a polypeptide corresponding to mature SoXy is present, clearly showing that export of SoXy in the wild-type strain had occurred in a strictly Tat-dependent manner. Another noteworthy finding is the observation that hardly any mature SoXy protein accumulated in the cellular fraction of the Tat⁺ wild-type strain (lane 3), indicating that SoXy is, after its Tat-dependent translocation across the cytoplasmic membrane and processing by signal peptidase, rapidly transported out of the intermembrane space across the mycolic acid-containing outer membrane into the supernatant. However, the mechanism of how proteins cross this additional permeability barrier is completely unknown so far (Bitter et al., 2009).Open in a separate windowFigure 3Transport of TorA–SoXy occurs in a strictly Tat-dependent manner. Plasmid pTorA–SoXy was transformed into C. glutamcium ATCC13032 (Tat⁺) and a C. glutamicum ΔTatAC mutant that lacks a functional Tat translocase (Meissner et al., 2007). As a control, the empty pEKEx2 expression vector was transformed into C. glutamicum ATCC13032 (Tat⁺). The respective strains were grown overnight in 5 ml of BHI medium (Difco) at 30°C. The cells were washed once with BHI and resuspended in 20 ml of fresh BHI medium containing 1 mM IPTG. After 6 h of further growth at 30°C, the cellular (C) and supernatant (S) fractions were prepared as described previously (Meissner et al., 2007). Samples of the C and S fractions were subjected to SDS-PAGE followed by immunoblotting using anti-SoXy antibodies as indicated at the top of the figure. Lanes 1 and 2: C. glutamicum ATCC13032 (pEKEx2); lanes 3 and 4: C. glutamicum ATCC13032 (pTorA–SoXy); lanes 5 and 6: C. glutamicum ΔTatAC (pTorA–SoXy). Asterisk: TorA–SoXy precursor; arrow: secreted SoXy protein. The positions of molecular mass markers (kDa) are indicated at the left margin of the figure.To the best of our knowledge, our results represent the first documented example of the successful secretion of a normally cytosolic, cofactor-containing protein via the Tat pathway in an active form into the culture supernatant of a recombinant expression host. Our results clearly show that, also for this biotechnologically very interesting class of proteins, a secretory production strategy can be a promising alternative to conventional intracellular expression strategies. Besides for SoXy and other FAD-containing carbohydrate oxidases, for which various applications are perceived by industry such as the in situ generation of hydrogen peroxide for bleaching and disinfection performance in technical applications, their use in the food and drink industry, as well as their use in diagnostic applications and carbohydrate biosynthesis processes (Oda and Hiraga, 1998; Murooka and Yamashita, 2001; van Hellemond et al., 2006; Heuts et al., 2007), a secretory production strategy might now be an attractive option also for biotechnologically relevant enzymes that are used as biocatalysts in chemo-enzymatic syntheses and that possess cofactors other than FAD, such as pyridoxal-5′-phosphate (PLP)-dependent ω-transaminases (Mathew and Yun, 2012) or various thiamin diphosphate (TDP)-dependent enzymes (Müller et al., 2009).     

Loss of TRAIL-Receptors Is a Recurrent Feature in Pancreatic Cancer and Determines the Prognosis of Patients with No Nodal Metastasis after Surgery

Introduction

Agonistic antibodies targeting TRAIL-receptors 1 and 2 (TRAIL-R1 and TRAIL-R2) are being developed as a novel therapeutic approach in cancer therapy including pancreatic cancer. However, the cellular distribution of these receptors in primary pancreatic cancer samples has not been sufficiently investigated and no study has yet addressed the issue of their prognostic significance in this tumor entity.

Aims and Methods

Applying tissue microarray (TMA) analysis, we performed an immunohistochemical assessment of TRAIL-receptors in surgical samples from 84 consecutive patients affected by pancreatic adenocarcinoma and in 26 additional selected specimens from patients with no lymph nodes metastasis at the time of surgery. The prognostic significance of membrane staining and staining intensity for TRAIL-receptors was evaluated.

Results

The fraction of pancreatic cancer samples with positive membrane staining for TRAIL-R1 and TRAIL-R2 was lower than that of cells from surrounding non-tumor tissues (TRAIL-R1: p<0.001, TRAIL-R2: p = 0.006). In addition, subgroup analyses showed that loss of membrane staining for TRAIL-R2 was associated with poorer prognosis in patients without nodal metastases (multivariate Cox regression analysis, Hazard Ratio: 0.44 [95% confidence interval: 0.22−0.87]; p = 0.019). In contrast, analysis of decoy receptors TRAIL-R3 and -R4 in tumor samples showed an exclusively cytoplasmatic staining pattern and no prognostic relevance.

Conclusion

This is a first report on the prognostic significance of TRAIL-receptors expression in pancreatic cancer showing that TRAIL-R2 might represent a prognostic marker for patients with early stage disease. In addition, our data suggest that loss of membrane-bound TRAIL-receptors could represent a molecular mechanism for therapeutic failure upon administration of TRAIL-receptors-targeting antibodies in pancreatic cancer. This hypothesis should be evaluated in future clinical trials.     

Saprolegnia strains isolated from river insects and amphipods are broad spectrum pathogens

Saprolegnia species are destructive pathogens to many aquatic organisms and are found in most parts of the world. Reports based on phylogenetic analysis suggest that Saprolegnia strains isolated from aquatic animals such as crustaceans and frogs are close to Saprolegnia strains isolated from infected fish or fish eggs and vice versa. However, it has often been assumed that host specificity occurs for each individual isolate or strain. Here we demonstrate that Saprolegnia spp. can have multiple hosts and are thus capable of infecting different aquatic organisms. Saprolegnia delica, Saprolegnia hypogyna, and 2 strains of Saprolegnia diclina were isolated from aquatic insects and amphipods while S. delica, Saprolegnia ferax, Pythium pachycaule, and a Pythium sp. were isolated from the water of a medium to fast flowing river. The ITS region of the rRNA gene was sequenced for all isolates. In challenge experiments, all four isolates from insects were found to be highly pathogenic to eggs of Atlantic salmon (Salmo salar) and embryos of the African clawed frog (Xenopus laevis). We found that Saprolegnia spp. isolated from salmon eggs were also able to successfully establish infection in nymphs of stonefly (Perla bipunctata) and embryos of X. laevis). These results suggest that Saprolegnia spp. are capable of infecting multiple hosts, which may give them an advantage during seasonal variation in their natural environments.     

The Value of In Vitro Diagnostic Testing in Medical Practice: A Status Report

BackgroundIn vitro diagnostic (IVD) investigations are indispensable for routine patient management. Appropriate testing allows early-stage interventions, reducing late-stage healthcare expenditure (HCE).AimTo investigate HCE on IVDs in two developed markets and to assess the perceived value of IVDs on clinical decision-making. Physician-perceived HCE on IVD was evaluated, as well as desired features of new diagnostic markers.MethodsPast and current HCE on IVD was calculated for the US and Germany. A total of 79 US/German oncologists and cardiologists were interviewed to assess the number of cases where: physicians ask for IVDs; IVDs are used for initial diagnosis, treatment monitoring, or post-treatment; and decision-making is based on an IVD test result. A sample of 201 US and German oncologists and cardiologists was questioned regarding the proportion of HCE they believed to be attributable to IVD testing. After disclosing the actual IVD HCE, the physician’s perception of the appropriateness of the amount was captured. Finally, the association between physician-rated impact of IVD on decision-making and perceived contribution of IVD expenditure on overall HCE was assessed.ResultsIVD costs account for 2.3% and 1.4% of total HCE in the US and Germany. Most physicians (81%) believed that the actual HCE on IVDs was >5%; 19% rated the spending correctly (0–4%, p<0.001). When informed of the actual amount, 64% of physicians rated this as appropriate (p<0.0001); 66% of decision-making was based on IVD. Significantly, more physicians asked for either additional clinical or combined clinical/health economic data than for the product (test/platform) alone (p<0.0001).ConclusionsOur results indicate a poor awareness of actual HCE on IVD, but a high attributable value of diagnostic procedures for patient management. New markers should deliver actionable and medically relevant information, to guide decision-making and foster improved patient outcomes.     

Tuberculosis Disease during Pregnancy and Treatment Outcomes in HIV-Infected and Uninfected Women at a Referral Hospital in Cape Town

BackgroundTuberculosis during pregnancy and treatment outcomes are poorly defined in high prevalence tuberculosis and HIV settings.MethodsA prospective cohort study of pregnant and postpartum women identified to be routinely on antituberculosis treatment was conducted at Tygerberg Hospital, Cape Town, South Africa, from January 2011 through December 2011. Maternal tuberculosis disease spectrum and tuberculosis-exposed newborns were characterized by maternal HIV status. Maternal tuberculosis treatment outcomes were documented and a multivariable regression model identified predictors of unfavourable tuberculosis treatment outcomes. Infant outcomes were also described.ResultsSeventy-four women with tuberculosis, 53 (72%) HIV-infected, were consecutively enrolled; 35 (47%) were diagnosed at delivery or postpartum and 22 (30%) of women reported previous antituberculosis treatment. HIV-infected women were 5.67 times more likely to have extrapulmonary tuberculosis (95% CI 1.18–27.25, p = 0.03). All 5 maternal deaths were amongst HIV-infected women. Birth outcomes were available for 75 newborns (2 sets of twins, missing data for 1 stillbirth). Of the 75 newborns, 49 (65%) were premature and 44 (59%) were low birth weight (LBW; <2500 grams). All 6 infants who died and the 4 stillbirths were born to HIV-infected women. Unfavourable tuberculosis treatment outcomes were documented in 33/74 (45%) women. Unfavourable maternal tuberculosis outcome was associated with delivery of LBW infants (OR 3.83; 95% CI 1.40–10.53, p = 0.009).ConclusionsA large number of pregnant women with tuberculosis presented at a provincial referral hospital. All maternal and infant deaths occurred in HIV-infected women and their newborns. Maternal tuberculosis treatment outcomes were poor.     

Accuracy of Non-Enhanced CT in Detecting Early Ischemic Edema Using Frequency Selective Non-Linear Blending

Purpose

Ischemic brain edema is subtle and hard to detect by computed tomography within the first hours of stroke onset. We hypothesize that non-enhanced CT (NECT) post-processing with frequency-selective non-linear blending (“best contrast”/BC) increases its accuracy in detecting edema and irreversible tissue damage (infarction).

Methods

We retrospectively analyzed the NECT scans of 76 consecutive patients with ischemic stroke (exclusively middle cerebral artery territory—MCA) before and after post-processing with BC both at baseline before reperfusion therapy and at follow-up (5.73±12.74 days after stroke onset) using the Alberta Stroke Program Early CT Score (ASPECTS). We assessed the differences in ASPECTS between unprocessed and post-processed images and calculated sensitivity, specificity, and predictive values of baseline NECT using follow-up CT serving as reference standard for brain infarction.

Results

NECT detected brain tissue hypoattenuation in 35 of 76 patients (46.1%). This number increased to 71 patients (93.4%) after post-processing with BC. Follow-up NECT confirmed brain infarctions in 65 patients (85.5%; p = 0.012). Post-processing increased the sensitivity of NECT for brain infarction from 35/65 (54%) to 65/65 (100%), decreased its specificity from 11/11 (100%) to 7/11 (64%), its positive predictive value (PPV) from 35/35 (100%) to 65/69 (94%) and increased its accuracy 46/76 (61%) to 72/76 (95%).

Conclusions

This post-hoc analysis suggests that post-processing of NECT with BC may increase its sensitivity for ischemic brain damage significantly.     

A new sauropodomorph ichnogenus from the Lower Jurassic of Sichuan,China fills a gap in the track record

A dinosaur tracksite in the Lower Jurassic Ziliujing Formation of Sichuan Province, China consists of a spectacular sub-vertical exposure, with multiple track-bearing levels and trackways showing parallel and bimodal orientations. Based on well-preserved material, the new ichnogenus and ichnospecies, Liujianpus shunan ichnogen. nov. ichnosp. nov. is erected to accommodate distinctive sauropodomorph trackways occurring in this assemblage. Liujianpus has a unique combination of features, some relating to the early Jurassic basal sauropodomorph (prosauropod in traditional usage) ichnogenus Otozoum, others to the sauropod ichnogenus Brontopodus. Despite such a mix of basal sauropodomorph- and sauropod-like features, the trackmaker of Liujianpus is likely a basal sauropodomorph. This identification is consistent with the occurrence of basal sauropodomorph skeletons from geographically and chronologically close localities. The other distinct morphotype from the tracksite is linked to a sauropod trackmaker. As such, the ichnofauna consisting of two distinct foot morphotypes reflects the diversity of sauropodomorph dinosaurs in the Early Jurassic of Asia.