150 Getting a grip on alpha-synuclein amyloid oligomers

Misfolding and aggregation of proteins into nanometer-scale fibrillar assemblies is a hallmark of many neurodegenerative diseases. Aggregation of the human alpha-synuclein protein is implicated in the etiology of Parkinson’s disease. A particularly relevant question is the role of early oligomeric aggregates of alpha-synuclein in modulating the dynamics of protein aggregation, and in the interactions with essential cellular components. However, very little is known about the molecular details of these aggregate species. For large protein aggregates, such as alpha-synuclein oligomers, it is very difficult to determine the number of monomers that form an oligomer using conventional techniques. We have developed a method that uses sub-stoichiometric labeling, that is, only a fraction of the monomers contains a fluorescent label, in combination with single-molecule photobleaching to determine the number of monomers per oligomer (Zijlstra et al., 2012). The number of bleaching steps gives the number of fluorescent labels per oligomer. Knowing the exact label density, that is, the fraction of labeled monomers at the start of the aggregation, we can correlate the number of fluorescent labels per oligomer to the total number of monomers. Using this method, we can determine the composition, probe the distribution in the number of monomers per oligomer, and investigate the influence of the fluorescent label on the aggregation process. For wild-type alpha-synuclein, we find no distribution in the number of monomers per oligomer and find a single, well-defined oligomeric species consisting of ～30 monomers per oligomer. On the other hand, for oligomers formed in the presence of dopamine, we find a distinctly bimodal distribution suggesting the existence of two populations of oligomeric species.     

Molecular dynamics simulations and density functional theory studies of NALMA and NAGMA dipeptides

Classical molecular dynamics (MD) simulations using fixed charged force field (AMBER ff03) and density functional theory method using the M05-2X/6-31G^?? level of theory have been used to investigate the plasticity of the hydrogen bond formed between dipeptides of N-Acetyl-Leucine-MethylAmide (NALMA), N-Acetyl-Glycine-MethylAmide (NAGMA), and vicinity of water molecules at temperature of 300?K. We have noticed that 2–3 water molecules contribute to change in the conformations of dipeptides NAGMA and NALMA. The self-assembly of 11 water molecules leads to the formation of water bridge at vicinity of the dipeptides and it constrain the conformations of dipeptides. We have found that the energy balance between breaking of the C?=?O…H–N H bonds and the formation of the C?=?O…H–O (wat) H bonds may be one of the determining factors to control the dynamics of the folding process of protein molecules.     

Development of cell-active non-peptidyl inhibitors of cysteine cathepsins

Cysteine cathepsins are an important class of enzymes that coordinate a variety of important cellular processes, and are implicated in various types of human diseases. However, small molecule inhibitors that are cell-permeable and non-peptidyl in nature are scarcely available. Herein the synthesis and development of sulfonyloxiranes as covalent inhibitors of cysteine cathepsins are reported. From a library of compounds, compound 5 is identified as a selective inhibitor of cysteine cathepsins. Live cell imaging and immunocytochemistry of metastatic human breast carcinoma MDA-MB-231 cells document the efficacy of compound 5 in inhibiting cysteine cathepsin activity in living cells. A cell-motility assay demonstrates that compound 5 is effective in mitigating the cell-migratory potential of highly metastatic breast carcinoma MDA-MB-231 cells.     

The Expression of IL6 and 21 in Crossbred Calves Upregulated by Inactivated Trivalent FMD Vaccine

Foot and mouth disease (FMD) is an economically important disease and a whole-virus inactivated trivalent virus vaccine is the mainstay for controlling the disease in India. The protective humoral immune response to FMD vaccination is a complex, but, tightly regulated process mediated by the interplay of interleukins (IL). Based on the specific role of IL6 and 21 in adaptive immune response, we hypothesized that inactivated trivalent FMD vaccine would stimulate IL6 and 21 expression in the circulating lymphocytes. The expressions of IL6 and 21 were assayed on 0, 28, 60, 90, and 120 d post-vaccination (DPV) by quantitative PCR (qPCR) with simultaneous assessment of FMDV antibody titer by liquid phase blocking ELISA. The results revealed that the peak expression of IL6 and 21 was on DPV 28 which correlated well with the FMDV antibody titer and plummeted to the prevaccination titer level by 60 DPV. As IL21 is the final effector of antibody production as compared to IL6, we investigated the expression of IL21 in calves that had protective titer (>1.8) with the unprotected group (<1.8). Expression of IL21 on 28 DPV was numerically higher in the protected than that of the unprotected group of calves.     

A collaborative framework for 3D alignment and classification of heterogeneous subvolumes in cryo-electron tomography

The limitation of using low electron doses in non-destructive cryo-electron tomography of biological specimens can be partially offset via averaging of aligned and structurally homogeneous subsets present in tomograms. This type of sub-volume averaging is especially challenging when multiple species are present. Here, we tackle the problem of conformational separation and alignment with a “collaborative” approach designed to reduce the effect of the “curse of dimensionality” encountered in standard pair-wise comparisons. Our new approach is based on using the nuclear norm as a collaborative similarity measure for alignment of sub-volumes, and by exploiting the presence of symmetry early in the processing. We provide a strict validation of this method by analyzing mixtures of intact simian immunodeficiency viruses SIV mac239 and SIV CP-MAC. Electron microscopic images of these two virus preparations are indistinguishable except for subtle differences in conformation of the envelope glycoproteins displayed on the surface of each virus particle. By using the nuclear norm-based, collaborative alignment method presented here, we demonstrate that the genetic identity of each virus particle present in the mixture can be assigned based solely on the structural information derived from single envelope glycoproteins displayed on the virus surface.     

Derivation of Neural Stem Cells from Human Adult Peripheral CD34+ Cells for an Autologous Model of Neuroinflammation

Proinflammatory factors from activated T cells inhibit neurogenesis in adult animal brain and cultured human fetal neural stem cells (NSC). However, the role of inhibition of neurogenesis in human neuroinflammatory diseases is still uncertain because of the difficulty in obtaining adult NSC from patients. Recent developments in cell reprogramming suggest that NSC may be derived directly from adult fibroblasts. We generated NSC from adult human peripheral CD34+ cells by transfecting the cells with Sendai virus constructs containing Sox2, Oct3/4, c-Myc and Klf4. The derived NSC could be differentiated to glial cells and action potential firing neurons. Co-culturing NSC with activated autologous T cells or treatment with recombinant granzyme B caused inhibition of neurogenesis as indicated by decreased NSC proliferation and neuronal differentiation. Thus, we have established a unique autologous in vitro model to study the pathophysiology of neuroinflammatory diseases that has potential for usage in personalized medicine.     

Bcl10 plays a divergent role in NK cell-mediated cytotoxicity and cytokine generation

Activating receptors such as NKG2D and Ly49D mediate a multitude of effector functions including cytotoxicity and cytokine generation in NK cells. However, specific signaling events that are responsible for the divergence of distinct effector functions have yet to be determined. In this study, we show that lack of caspase recruitment domain-containing protein Bcl10 significantly affected receptor-mediated cytokine and chemokine generation, but not cytotoxicity against tumor cells representing "missing-self" or "induced-self." Lack of Bcl10 completely abrogated the generation of GM-CSF and chemokines and it significantly reduced the generation of IFN-gamma (>75%) in NK cells. Commitment, development, and terminal maturation of NK cells were largely unaffected in the absence of Bcl10. Although IL-2-activated NK cells could mediate cytotoxicity to the full extent, the ability of the freshly isolated NK cells to mediate cytotoxicity was somewhat reduced. Therefore, we conclude that the Carma1-Bcl10-Malt1 signaling axis is critical for cytokine and chemokine generation, although it is dispensable for cytotoxic granule release depending on the activation state of NK cells. These results indicate that Bcl10 represents an exclusive "molecular switch" that links the upstream receptor-mediated signaling to cytokine and chemokine generations.