Nanoscale dynamics of cellulose digestion by the cellobiohydrolase TrCel7A

Understanding the mechanism by which cellulases from bacteria, fungi, and protozoans catalyze the digestion of lignocellulose is important for developing cost-effective strategies for bioethanol production. Cel7A from the fungus Trichoderma reesei is a model exoglucanase that degrades cellulose strands from their reducing ends by processively cleaving individual cellobiose units. Despite being one of the most studied cellulases, the binding and hydrolysis mechanisms of Cel7A are still debated. Here, we used single-molecule tracking to analyze the dynamics of 11,116 quantum dot-labeled TrCel7A molecules binding to and moving processively along immobilized cellulose. Individual enzyme molecules were localized with a spatial precision of a few nanometers and followed for hundreds of seconds. Most enzyme molecules bound to cellulose in a static state and dissociated without detectable movement, whereas a minority of molecules moved processively for an average distance of 39 nm at an average speed of 3.2 nm/s. These data were integrated into a three-state model in which TrCel7A molecules can bind from solution into either static or processive states and can reversibly switch between states before dissociating. From these results, we conclude that the rate-limiting step for cellulose degradation by Cel7A is the transition out of the static state, either by dissociation from the cellulose surface or by initiation of a processive run. Thus, accelerating the transition of Cel7A out of its static state is a potential avenue for improving cellulase efficiency.     

Advances in the Chromosome-Centric Human Proteome Project: looking to the future

Introduction: The mission of the Chromosome-Centric Human Proteome Project (C-HPP), is to map and annotate the entire predicted human protein set (~20,000 proteins) encoded by each chromosome. The initial steps of the project are focused on ‘missing proteins (MPs)’, which lacked documented evidence for existence at protein level. In addition to remaining 2,579 MPs, we also target those annotated proteins having unknown functions, uPE1 proteins, alternative splice isoforms and post-translational modifications. We also consider how to investigate various protein functions involved in cis-regulatory phenomena, amplicons lncRNAs and smORFs.

Areas covered: We will cover the scope, historic background, progress, challenges and future prospects of C-HPP. This review also addresses the question of how we can best improve the methodological approaches, select the optimal biological samples, and recommend stringent protocols for the identification and characterization of MPs. A new strategy for functional analysis of some of those annotated proteins having unknown function will also be discussed.

Expert commentary: If the project moves well by reshaping the original goals, the current working modules and team work in the proposed extended planning period, it is anticipated that a progressively more detailed draft of an accurate chromosome-based proteome map will become available with functional information.     

CD134 as target for specific drug delivery to auto-aggressive CD4<Superscript>+</Superscript>T cells in adjuvant arthritis

T cells have an important role during the development of autoimmune diseases. In adjuvant arthritis, a model for rheumatoid arthritis, we found that the percentage of CD4⁺ T cells expressing the activation marker CD134 (OX40 antigen) was elevated before disease onset. Moreover, these CD134⁺ T cells showed a specific proliferative response to the disease-associated epitope of mycobacterial heat shock protein 60, indicating that this subset contains auto-aggressive T cells. We studied the usefulness of CD134 as a molecular target for immune intervention in arthritis by using liposomes coated with a CD134-directed monoclonal antibody as a drug targeting system. Injection of anti-CD134 liposomes subcutaneously in the hind paws of pre-arthritic rats resulted in targeting of the majority of CD4⁺CD134⁺ T cells in the popliteal lymph nodes. Furthermore, we showed that anti-CD134 liposomes bound to activated T cells were not internalized. However, drug delivery by these liposomes could be established by loading anti-CD134 liposomes with the dipalmitate-derivatized cytostatic agent 5'-fluorodeoxyuridine. These liposomes specifically inhibited the proliferation of activated CD134⁺ T cells in vitro, and treatment with anti-CD134 liposomes containing 5'-fluorodeoxyuridine resulted in the amelioration of adjuvant arthritis. Thus, CD134 can be used as a marker for auto-aggressive CD4⁺ T cells early in arthritis, and specific liposomal targeting of drugs to these cells via CD134 can be employed to downregulate disease development.     

Analysis of dynamic changes in the proteome of a Bcl-XL overexpressing Chinese hamster ovary cell culture during exponential and stationary phases

Mammalian cell cultures used for biopharmaceutical production undergo various dynamic biological changes over time, including the transition of cells from an exponential growth phase to a stationary phase during cell culture. To better understand the dynamic aspects of cell culture, a quantitative proteomics approach was used to identify dynamic trends in protein expression over the course of a Chinese hamster ovary (CHO) cell culture for the production of a recombinant monoclonal antibody and overexpressing the antiapoptotic gene Bcl-xl. Samples were analyzed using a method incorporating iTRAQ labeling, two-dimensional LC/MS, and linear regression calculations to identify significant dynamic trends in protein abundance. Using this approach, 59 proteins were identified with significant temporal changes in expression. Pathway analysis tools were used to identify a putative network of proteins associated with cell growth and apoptosis. Among the differentially expressed proteins were molecular chaperones and isomerases, such as GRP78 and PDI, and reported cell growth markers MCM2 and MCM5. In addition, two proteins with growth-regulating properties, transglutaminase-2 and clusterin, were identified. These proteins are associated with tumor proliferation and apoptosis and were observed to be expressed at relatively high levels during stationary phase, which was confirmed by western blotting. The proteomic methodology described here provides a dynamic view of protein expression throughout a CHO fed-batch cell culture, which may be useful for further elucidating the biological processes driving mammalian cell culture performance.     

Synthesis of peptide labelled with p-iodophenylalanine which corresponds to the first amphipathic region of apolipoprotein C-I

The amino terminal fragment (1-15) of apolipoprotein C-1, H-Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe14-Gly was prepared by the solid phase method. However, the phenylalanine residue at position 14 was replaced with p-iodophenylalanine in the chemical synthesis. The preparation of t-butyloxy-carbonyl-p-iodophenylalanine is described. After completion of the synthesis, the product was deprotected and cleaved from the resin with liquid HF. The peptide was purified by gel filtration on Sephadex G-25 and preparative high performance liquid chromatography. The purified material was shown to be homogeneous by amino acid analytical data and by chromatography in three different analytical reversed phase HPLC systems. The peptide was then labelled by the chloroamine-T procedure and good incorporation of 125I was obtained. After purification of the product by gel filtration on Biogel P-2, the labelled pentadecapeptide was tested for ability to bind to Very Low Density Lipoproteins (VLDL) in the following manner: VLDL was isolated from normolipemic serum by ultracentrifugal flotation and 100-microliter samples were incubated with labelled material dissolved in 200 microliter of 0.5 M NaCl, 0.02 M sodium phosphate buffer, pH 7.4 at 37 degrees for 30 min. The VLDL fraction was again isolated by ultracentrifugal flotation and the incorporation of radioactivity into the lipoprotein was measured. Under these conditions, a sample of [3H]-native apolipoprotein C-I was incorporated to an extent of 83% of the total sample, while the [125I]-pentadecapeptide exhibited an incorporation of 8.7%.