A structural study of Hypocrea jecorina Cel5A

Interest in generating lignocellulosic biofuels through enzymatic hydrolysis continues to rise as nonrenewable fossil fuels are depleted. The high cost of producing cellulases, hydrolytic enzymes that cleave cellulose into fermentable sugars, currently hinders economically viable biofuel production. Here, we report the crystal structure of a prevalent endoglucanase in the biofuels industry, Cel5A from the filamentous fungus Hypocrea jecorina. The structure reveals a general fold resembling that of the closest homolog with a high-resolution structure, Cel5A from Thermoascus aurantiacus. Consistent with previously described endoglucanase structures, the H. jecorina Cel5A active site contains a primarily hydrophobic substrate binding groove and a series of hydrogen bond networks surrounding two catalytic glutamates. The reported structure, however, demonstrates stark differences between side-chain identity, loop regions, and the number of disulfides. Such structural information may aid efforts to improve the stability of this protein for industrial use while maintaining enzymatic activity through revealing nonessential and immutable regions

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Combinatorial Recombination of Gene Fragments to Construct a Library of Chimeras

Recombination of distantly related and nonrelated genes is difficult using traditional PCR‐based techniques, and truncation‐based methods result in a large proportion of nonviable sequences due to frame shifts, deletions, and insertions. This unit describes a method for creating libraries of chimeras through combinatorial assembly of gene fragments. It allows the experimenter to recombine genes of any identity and to select the sites where recombination takes place. Combinatorial recombination is achieved by generating gene fragments with specific overhangs, or sticky ends. The overhangs permit the fragments to be ligated in the correct order while allowing independent assortment of blocks with identical overhangs. Genes of any identity can be recombined so long as they share 3 to 5 base pairs of identity at the desired recombination sites. Simple adaptations of the method allow incorporation of specific gene fragments

Function of a Mycobacterial Major Facilitator Superfamily Pump Requires a Membrane-Associated Lipoprotein▿

The lprG-Rv1410c operon is critical for the survival of Mycobacterium tuberculosis during infection, but very little is known about the functions of its proteins. LprG is a lipoprotein, and Rv1410c encodes the major facilitator superfamily small molecule transporter P55. P55 likely exports small molecules outside of the bacterial cell, but the function of LprG is unclear. A deletion of the homologous operon in Mycobacterium smegmatis is more susceptible to ethidium bromide, and drug resistance is restored by the intact operon from M. tuberculosis. The multidrug resistance pump inhibitor reserpine inhibits resistance to ethidium bromide in both wild-type M. smegmatis and the complemented mutant, suggesting that P55-mediated transport is responsible for drug resistance and that ethidium bromide is a novel substrate for P55. In addition to hypersensitivity to ethidium bromide, cells that lack the lprG-Rv1410c operon display abnormal colony morphology and are defective for sliding motility, properties that suggest an alteration of cell wall composition. Strikingly, both ethidium bromide transport and normal cell surface properties require functional P55 and LprG, as neither alone is sufficient to restore function to the deletion mutant. Thus, P55 requires the cell surface lipoprotein for normal function

A diverse set of family 48 bacterial glycoside hydrolase cellulases created by structure-guided recombination

Sequence diversity within a family of functional enzymes provides a platform for elucidating structure–function relationships and for protein engineering to improve properties important for applications. Access to nature's vast sequence diversity is often limited by the fact that only a few enzymes have been characterized in a given family. Here, we recombined the catalytic domains of three glycoside hydrolase family 48 bacterial cellulases (Cel48; EC 3.2.1.176) – Clostridium cellulolyticum CelF, Clostridium stercorarium CelY, and Clostridium thermocellum CelS – to create a diverse library of Cel48 enzymes with an average of 106 mutations from the closest native enzyme. Within this set, we found large variations in properties such as the functional temperature range, stability, and specific activity on crystalline cellulose. We showed that functional status and stability were predictable from simple linear models of the sequence–property data: recombined protein fragments contributed additively to these properties in a given chimera. Using this, we correctly predicted sequences that were as stable as any of the native Cel48 enzymes described to date. The characterization of 60 active Cel48 chimeras expands the number of characterized Cel48 enzymes from 13 to 73. Our work illustrates the role that structure-guided recombination can play in helping to identify sequence–function relationships within a family of enzymes by supplementing natural diversity with synthetic diversity

C-erbB3 and c-erbB4 expression is a feature of the endocrine responsive phenotype in clinical breast cancer

We examined c-erbB3 and c-erbB4 mRNA expression in 47 primary breast cancer samples by simultaneous RT-PCR and have investigated correlations between these parameters and the expression of both ER and EGFR mRNA and protein as measured by RT-PCR and ICA and with Ki67 immunostaining. A direct association was found between c-erbB3 and c-erbB4 mRNA and ER marker status measured by either RT-PCR (c-erbB3 P=0.0003; c-erbB4 P=0.02) or ICA (c-erbB-3 P=0.002; c-erbB4 P=0.01). Inverse associations were seen between c-erbB3 and c-erbB4 mRNA marker status and EGFR membrane protein (c-erbB3: P=0.003; c-erbB4: P=0.003) and mRNA (c-erbB4: P=0.009) status. These associations were reinforced by Spearman Rank Correlation Tests. A significant relationship was seen between Ki67 and c-erbB4 mRNA status and level. Measurements of c-erbB3 protein levels in tumour samples removed from a further 89 patients of known response to endocrine therapy: (i) confirmed the relationship between c-erbB3 and ER and (ii) identified that patients whose ER positive tumours expressed high levels of c-erbB3 were most likely to benefit from endocrine measures. A non-significant trend was recorded between c-erbB3 levels and Ki67 immunostaining. These results clearly demonstrate that increased c-erbB3 and c-erbB4 expression appears to be associated with the prognostically-favourable ER phenotype

Grey matter differences in bipolar disorder: a meta-analysis of voxel-based morphometry studies

OBJECTIVE: Several neuroimaging studies have reported structural brain differences in bipolar disorder using automated methods. While these studies have several advantages over those using region of interest techniques, no study has yet estimated a summary effect size or tested for between-study heterogeneity. We sought to address this issue using meta-analytic techniques applied for the first time in bipolar disorder at the level of the individual voxel. METHODS: A systematic review identified 16 voxel-based morphometry (VBM) studies comparing individuals with bipolar disorder with unaffected controls, of which eight were included in the meta-analysis. In order to take account of heterogeneity, summary effect sizes were computed using a random-effects model with appropriate correction for multiple testing. RESULTS: Compared with controls, subjects with bipolar disorder had reduced grey matter in a single cluster encompassing the right ventral prefrontal cortex, insula, temporal cortex, and claustrum. Study heterogeneity was widespread throughout the brain; though the significant cluster of grey matter reduction remained once these extraneous voxels had been removed. We found no evidence of publication bias (Eggers p = 0.63). CONCLUSIONS: Bipolar disorder is consistently associated with reductions in right prefrontal and temporal lobe grey matter. Reductions elsewhere may be obscured by clinical and methodological heterogeneity