A streamlined, automated protocol for the production of milligram quantities of untagged recombinant rat lactate dehydrogenase A using ÄKTAxpress™

We developed an efficient, automated 2-step purification protocol for the production of milligram quantities of untagged recombinant rat lactate dehydrogenase A (rLDHA) from E. coli, using the ÄKTAxpress™ chromatography system. Cation exchange followed by size exclusion results in average final purity in excess of 93% and yields ~ 14 milligrams per 50 ml of original cell culture in EnPresso B media, in under 8 hrs, including all primary sample processing and column equilibration steps. The protein is highly active and coherent biophysically and a viable alternative to the more problematic human homolog for structural and ligand-binding studies; an apo structure of untagged rLDHA was solved to a resolution 2.29 Å (PDB ID 5ES3). Our automated methodology uses generic commercially available pre-packed columns and simple buffers, and represents a robust standard method for the production of milligram amounts of untagged rLDHA, facilitating a novel fragment screening approach for new inhibitors

Mechanical Functions of the Endothelial Glycocalyx

The first part of this thesis is dedicated to how endothelial cells (ECs) produce nitric oxide (NO) due to mechanical perturbations, a process known as mechanotransduction. NO is perhaps one of the most important protective molecules in the body and is responsible for vasorelaxation, which staves off high blood pressure. Although this field has been heavily researched over the past ten years a clear picture of the underlying mechanisms that allow for this affect to take place have yet to be elucidated. Based on previous research endeavors it has been shown that the gel like carbohydrate rich layer found on the surface of ECs is a key player in NO production when ECs are exposed to shear stress due to flow. This layer is known as the endothelial surface glycocalyx (ESG). The ESG contains the long oligosaccharide chain heparan sulfate (HS), that when removed will prevent ECs producing NO under flow conditions. Also, if the protein glypican-1 that anchors HS to the EC surface is knocked out, NO will not be produced under flow conditions. Furthermore, other studies have shown that ECs will not produce NO when transient receptor potential (TRP) channels are inhibited. These channels are mechanically activated, ion soluble and known to allow Ca2+ to enter ECs, a molecule that allows the NO production cascade to begin. Therefore, the primary question that remained unanswered until now was how the HS-glypican-1 complex mechanically activates TRP channels to begin the NO production cascade.The big picture problem that knowledge of this pathway may improve is treating diseases in which the ESG is disrupted. These diseases, such as diabetes and arteriosclerosis go hand in glove with reduced NO production which impairs vasorelaxation, leading to high blood pressure. However, the problem was that, until now, there has been no way to investigate the aforementioned question experimentally. Our solution to this was to utilize an atomic force microscope (AFM) to apply force to the surface of ECs, targeting the ESG, to induce NO production.An AFM is a highly sensitive tool that can provide minute mechanical forces to a cell via a micro cantilever. In chapter 3 we coated the AFM probe in ployLysine, an amino acid that has a strong positive charge. With this charge we were able to mechanically stimulate the surface of an EC due to the ESG’s strong negative charge. When the ESG was perturbed by the AFM probe NO was produced in the EC. When HS was removed NO was no longer produced after mechanical stimulation via AFM. Moreover, when the TRP channel TRPP2 was inhibited, NO was not produced. A publication of this method can be found in the American Journal of Physiology (doi:10.1152/ajpcell.00288.2015). From this we have concluded that the ESG component HS is placing mechanical stress on TRP channels, such as TRPP2, causing them to allow Ca2+ into the EC thus beginning the NO production cascade.After this time of initial discovery we focused on developing a new technique that will allow for the mechanical stimulation of HS or glypican-1 only on a fully intact ESG. Therefore, we can target the affects that HS or glypican-1 have on NO production directly, not simply infer what they do after HS is removed. This path was evaluated in chapter 4. It was the first experimental exploration to demonstrate that the affect witnessed in my first publication was not due to ESG damage after HS removal. To attain this goal we coated the AFM probes in glypican-1 antibodies to achieve specificity. The results allow us to conclude that applying mechanical force to glypican-1 will result in rapid NO production. Moreover, this NO production can be completely inhibited by blocking TRPC channels and reduced by blocking TRPP channels. Together, our results demonstrated that the molecular mechanism of rapid NO production is a result of glypican-1creating mechanical tension in the cell membrane, activating TRPC and TRPP channels.An additional chapter was written about the ESG’s affect in inflammation. This work involved using an AFM-based single-cell adhesion assay where we directly quantified the detachment force and work perpendicular to the cell membrane of a leukocyte from a human umbilical cord vein endothelial cell (HUVEC). To perform this delicate procedure we again turned to the use of a ployLysine coated probe. This allowed us to pick up an individual K562 leukocyte with the AFM probe. After doing this the K562 was brought over a single HUVEC for adhesion testing. This was performed with an intact ESG, or with the major ESG components, HS and/or hyaluronic acid (HA) removed. For the resting HUVECs, when HS and/or HA were removed, the detachment force and work increased. For the HUVECs activated by inflammatory cytokine tumor necrosis factor alpha, we observed increases in the detachment force and work compared to the resting HUVECs. Furthermore, under inflammatory conditions, removal of HS and/or HA resulted in significant decreases in the detachment force and work. The results demonstrated that the ESG layer serves a dual function: (1) on resting endothelium, it prevents leukocyte adhesion, and (2) under inflammatory conditions, it participates in endothelial-leukocyte interactions with molecules such as selectins. A publication on this work can be found in the BMES journal. (10.1007/s12195-016-0463-6).Lastly we performed an analysis on the reliability of AFM nanoindentation-derived measurements of cell mechanics. AFM is an experimental technique that is often used for measuring the mechanical properties of cells and other soft materials. Despite its widespread adoption as a biophysical assay, no universal standards have been adopted for the technique. This has potentially caused the problem of irreproducible results when scientists utilize this technique. The purpose of this study was to assess the accuracy and repeatability of AFM-derived cell stiffness measurements. Therefore, we conducted a series of experiments on ASPC-1 cells (A human pancreatic cancer cell line) to compare a variety of conditions that may be leading to this inaccuracy. The conditions that observed were the following: conical vs. spherical AFM tips, nuclear vs. peripheral indentation locations, multiple piezo actuation speeds and multiple indentation forces. We then quantified apparent cell stiffness using classical contact mechanics with the Hertz model for the spherical tip and Sneddon model for the conical tip. From this we could draw several significant conclusions. The most important is that across experimental conditions, cells appeared to be stiffer when probed with conical tips compared to spherical tips. Moreover, there were no differences in apparent cell stiffness based on AFM actuation speed or indentation force. We have thus concluded that AFM remains a valuable technique for probing the mechanical properties of cells, but greater consensus can be achieved through adoption of standardized methods

Endothelial Glycocalyx--The Battleground for Complications of Sepsis and Kidney Injury

After briefly discussing endothelial glycocalyx and its role in vascular physiology and renal disease, this overview focuses on its degradation very early in the course of microbial sepsis. We describe our recently proposed mechanism for glycocalyx degradation induced by exocytosis of lysosome-related organelles and release of their cargo. Notably, an intermediate in nitric oxide synthesis, NG-hydroxy-l-arginine, shows efficacy in curtailing exocytosis of these organelles and improvement in animal survival. These data not only depict a novel mechanism responsible for very early glycocalyx degradation, but may also outline a potential preventive therapy. The second issue discussed in this article is related to the therapeutic acceleration of restoration of already degraded endothelial glycocalyx. Here, using as an example our recent findings obtained with sulodexide, we illustrate the importance of the expedited repair of degraded endothelial glycocalyx for the survival of animals with severe sepsis. These two focal points of the review on glycocalyx may not only have broader disease applicability, but they may also provide additional evidence to buttress the idea of the importance of endothelial glycocalyx and its maintenance and repair in the prevention and treatment of an array of renal and nonrenal diseases

PD-1 signaling uncovers a pathogenic subset of T cells in inflammatory arthritis

Abstract Background PD-1 is an immune checkpoint on T cells, and interventions to block this receptor result in T cell activation and enhanced immune response to tumors and pathogens. Reciprocally, despite a decade of research, approaches to treat autoimmunity with PD-1 agonists have only had limited successful. To resolve this, new methods must be developed to augment PD-1 function beyond engaging the receptor. Methods We conducted a flow cytometry analysis of T cells isolated from the peripheral blood and synovial fluid of patients with rheumatoid arthritis. In addition, we performed a genome-wide CRISPR/Cas9 screen to identify genes associated with PD-1 signaling. We further analyzed genes involved in PD-1 signaling using publicly available bulk and single-cell RNA sequencing datasets. Results Our screen confirmed known regulators in proximal PD-1 signaling and, importantly, identified an additional 1112 unique genes related to PD-1 ability to inhibit T cell functions. These genes were strongly associated with the response of cancer patients to PD-1 blockades and with high tumor immune dysfunction and exclusion scores, confirming their role downstream of PD-1. Functional annotation revealed that the most significant genes uncovered were those associated with known immune regulation processes. Remarkably, these genes were considerably downregulated in T cells isolated from patients with inflammatory arthritis, supporting their overall inhibitory functions. A study of rheumatoid arthritis single-cell RNA sequencing data demonstrated that five genes, KLRG1, CRTAM, SLAMF7, PTPN2, and KLRD1, were downregulated in activated and effector T cells isolated from synovial fluids. Backgating these genes to canonical cytotoxic T cell signatures revealed PD-1+ HLA-DRHIGH KLRG1LOW T cells as a novel inflammatory subset of T cells. Conclusions We concluded that PD-1+ HLA-DRHIGH KLRG1LOW T cells are a potential target for future PD-1 agonists to treat inflammatory diseases. Our study uncovers new genes associated with PD-1 downstream functions and, therefore, provides a comprehensive resource for additional studies that are much needed to characterize the role of PD-1 in the synovial subset of T cells

Cryo-EM reveals an unprecedented binding site for NaV1.7 inhibitors enabling rational design of potent hybrid inhibitors

The voltage-gated sodium (NaV) channel NaV1.7 has been identified as a potential novel analgesic target due to its involvement in human pain syndromes. However, clinically available NaV channel-blocking drugs are not selective among the nine NaV channel subtypes, NaV1.1–NaV1.9. Moreover, the two currently known classes of NaV1.7 subtype-selective inhibitors (aryl- and acylsulfonamides) have undesirable characteristics that may limit their development. To this point understanding of the structure–activity relationships of the acylsulfonamide class of NaV1.7 inhibitors, exemplified by the clinical development candidate GDC-0310, has been based solely on a single co-crystal structure of an arylsulfonamide inhibitor bound to voltage-sensing domain 4 (VSD4). To advance inhibitor design targeting the NaV1.7 channel, we pursued high-resolution ligand-bound NaV1.7-VSD4 structures using cryogenic electron microscopy (cryo-EM). Here, we report that GDC-0310 engages the NaV1.7-VSD4 through an unexpected binding mode orthogonal to the arylsulfonamide inhibitor class binding pose, which identifies a previously unknown ligand binding site in NaV channels. This finding enabled the design of a novel hybrid inhibitor series that bridges the aryl- and acylsulfonamide binding pockets and allows for the generation of molecules with substantially differentiated structures and properties. Overall, our study highlights the power of cryo-EM methods to pursue challenging drug targets using iterative and high-resolution structure-guided inhibitor design. This work also underscores an important role of the membrane bilayer in the optimization of selective NaV channel modulators targeting VSD4