Search

Immune mediated graft loss still represents a major risk to transplant recipients. Creative approaches to immunosuppression that exploit the recipient's own alloregulatory mechanisms could reduce the need for pharmacologic immunosuppression and potentially induce immune tolerance. In the process of studying recipient derived myeloid derived suppressor cells (MDSCs), we identified key alloregulatory MDSC mechanisms, mediated by isolatable proteins IL-4, IL-34, and IL-10. We sought to purify these proteins and fuse them for subsequent infusion into transplant recipients as a means of inducing an alloregulatory response. In this introductory investigation, we leveraged molecular engineering technology to create a fusion protein (FP) of three cytokine coding sequences of IL-4, IL-34, and IL-10 and demonstrated their expressions by Western Blot analysis. Following purification, we tested whether FP IL-4/IL-34/IL-10 (FP1) can protect heart transplant allografts. Injection of FP1 significantly prolonged allogeneic cardiac graft survival in a dose-dependent fashion and the increase of graft survival time exceeded survival attributable to IL-34 alone. In vitro, MDSCs cells were expanded by FP1 treatment. However, FP1 did not directly inhibit T cell proliferation in vitro. In conclusion, newly developed FP1 improves the graft survival in cardiac transplantation mouse model. Significant additional work to optimize FP1 or include other novel proteins could supplement current treatment options for transplant patients.

Targeting Clostridium difficile infection is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent C. difficile strains often have a binary toxin termed the C. difficile toxin, in addition to the enterotoxins TsdA and TsdB. The C. difficile toxin has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of singleparticle cryoelectron microscopy, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, 2 di-heptamer structures for activated CDTb (1.0 MDa) were solved at atomic resolution, including a symmetric (SymCDTb; 3.14 Å) and an asymmetric form (AsymCDTb; 2.84 Å). Roles played by 2 receptor-binding domains of activated CDTb were of particular interest since the receptorbinding domain 1 lacks sequence homology to any other known toxin, and the receptor-binding domain 2 is completely absent in other well-studied heptameric toxins (i.e., anthrax). For AsymCDTb, a Ca2+ binding site was discovered in the first receptor-binding domain that is important for its stability, and the second receptor-binding domain was found to be critical for host cell toxicity and the diheptamer fold for both forms of activated CDTb. Together, these studies represent a starting point for developing structure-based drug-design strategies to target the most severe strains of C. difficile. [ABSTRACT FROM AUTHOR]

Vitamin A is a necessary nutrient for all mammals, and it is required for the transcription of many genes and vital for vision. While fasting, the vitamin A alcohol form (Retinol) from storage in the liver is mobilized and transported through the bloodstream while bound to retinol binding protein (RBP). Details of how exactly vitamin A is released from RBP and taken into the cells are still unclear. As part of the effort to elucidate the specifics of this process, single-particle cryo-electron microscopy structural studies of STRA6 (the RBP receptor 75-kDa transmembrane receptor protein) were recently reported by Chen et al. (Science, 10.1126/science.aad8266, 2016). Interestingly, STRA6 from zebrafish was shown to be a stable dimer and bound to calmodulin (CaM), forming a 180-kDa complex. The topology of the STRA6 complex includes 18 transmembrane helices (nine per protomer) and two long horizontal intramembrane helices interacting at the dimer core (Chen et al., in Science, 10.1126/science.aad8266, 2016). CaM was shown to interact with three regions of STRA6, termed CaMBP1, CaMBP2, and CaMBP3, with the most extensive interactions involving CaMBP2. To further our understanding of Ca2+-dependence of CaM-STRA6 complex formation, studies of the structure and dynamic properties of the CaMBP2–CaM complex were initiated. For this, the 1HN, 13C, and 15N backbone resonance assignments of the 148 amino acid Ca2+-bound calmodulin protein bound to the 27-residue CaMBP2 peptide derived from STRA6 were completed here using heteronuclear multidimensional NMR spectroscopy. [ABSTRACT FROM AUTHOR]

The application of the different alanine methyl groups as the NMR probes for the study of molecular structure and dynamics in high-molecular-weight proteins is demonstrated. These groups are shown to be extremely efficient and accurate for conducting such studies.

NMR method is developed for high-resolution, accurate measurements of methyl [super 1][H.sub.m]-[super 13][C.sub.m] ([super 1][D.sub.CH]) and [super 13][C.sub.m]-[super 13]C ([super 1][D.sub.CC]) residual dipolar couplings (RDCs) in ILV-methyl-protonated high-molecular-weight proteins. The studies have shown that the ratios of [super 1][D.sub.CH] and [super 1][D.sub.cc] couplings are independent of methyl axis dynamics and the residual alignment.

The article presents two different approaches that can be used for the determination of the quadrupolar coupling constants of [alpha] deuterons. The presence of the various [C.sup.[alpha]]-[H.sup.[alpha]]-O=C hydrogen bonds in these deuterons is also demonstrated.

The studies about gpW, a natural 62-residue [alpha] + [beta] protein that folds slowly in a two-state fashion within microseconds is presented. The results obtained along with the complex functional role observed in bacteriophage [lambda] assembly revealed that gpW is engineered to fold downhill by natural selection.

A variable-barrier model and an Ising-like model are used to estimate the barrier height from the calorimeter data. The free-energy barrier to folding is sufficiently low.