Molecular mechanisms of heterogeneous oligomerization of huntingtin proteins

There is still no successful strategy to treat Huntington's disease, an inherited autosomal disorder associated with the aggregation of mutated forms of the huntingtin protein containing polyglutamine tracts with more than 36 repeats. Recent experimental evidence is challenging the conventional view of the disease by revealing transcellular transfer of mutated huntingtin proteins which are able to seed oligomers involving wild type forms of the protein. Here we decipher the molecular mechanism of this unconventional heterogeneous oligomerization by performing discrete molecular dynamics simulations. We identify the most probable oligomer conformations and the molecular regions that can be targeted to destabilize them. Our computational findings are complemented experimentally by fluorescence-lifetime imaging microscopy/fluorescence resonance energy transfer (FLIM-FRET) of cells co-transfected with huntingtin proteins containing short and large polyglutamine tracts. Our work clarifies the structural features responsible for heterogeneous huntingtin aggregation with possible implications to contrast the prion-like spreading of Huntington's disease

Modifications to the framework regions eliminate chimeric antigen receptor tonic signaling

Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. Here, we report that CAR tonic signaling is caused by the intrinsic instability of the mAb single-chain variable fragment (scFv) to promote self-aggregation and signaling via the CD3z chain incorporated into the CAR construct. This phenomenon was detected in a CAR encoding either CD28 or 4-1BB costimulatory endodomains. Instability of the scFv was caused by specific amino acids within the framework regions (FWR) that can be identified by computational modeling. Substitutions of the amino acids causing instability, or humanization of the FWRs, corrected tonic signaling of the CAR, without modifying antigen specificity, and enhanced the antitumor effects of CAR-T cells. Overall, we demonstrated that tonic signaling of CAR-T cells is determined by the molecular instability of the scFv and that computational analyses of the scFv can be implemented to correct the scFv instability in CAR-T cells with either CD28 or 4-1BB costimulation

Modulation of the mitochondrial voltage dependent anion channel (VDAC) by curcumin

Voltage dependent anion channel (VDAC) of mitochondria plays a crucial role in apoptosis. Human VDAC-1, reconstituted in planar lipid bilayer showed reduced conductance when treated with curcumin. Curcumin interacts with residues in the alpha helical N-terminus of VDAC and in the channel wall, as revealed by molecular docking, followed by mutational analysis. N-terminus mimicking peptide showed conformational changes in circular dichroism, upon curcumin treatment. We propose that the interaction of curcumin with amino acids in N-terminus and in channel wall fixes the alpha helix in closed conformation. This restricts its movement which is required for the opening of the channel. (C) 2014 Elsevier B.V. All rights reserved

Naturally Occurring Variants of the Dysglycemic Peptide Pancreastatin DIFFERENTIAL POTENCIES FOR MULTIPLE CELLULAR FUNCTIONS AND STRUCTURE-FUNCTION CORRELATION

Pancreastatin (PST), a chromogranin A-derived peptide, is a potent physiological inhibitor of glucose-induced insulin secretion. PST also triggers glycogenolysis in liver and reduces glucose uptake in adipocytes and hepatocytes. Here, we probed for genetic variations in PST sequence and identified two variants within its functionally important carboxyl terminus domain: E287K and G297S. To understand functional implications of these amino acid substitutions, we tested the effects of wild-type (PST-WT), PST-287K, and PST-297S peptides on various cellular processes/events. The rank order of efficacy to inhibit insulin-stimulated glucose uptake was: PST-297S > PST-287K > PST-WT. The PST peptides also displayed the same order of efficacy for enhancing intracellular nitric oxide and Ca2+ levels in various cell types. In addition, PST peptides activated gluconeogenic genes in the following order: PST-297S approximate to PST-287K > PST-WT. Consistent with these in vitro results, the common PST variant allele Ser-297 was associated with significantly higher (by approximate to 17 mg/dl, as compared with the wild-type Gly-297 allele) plasma glucose level in our study population (n = 410). Molecular modeling and molecular dynamics simulations predicted the following rank order of -helical content: PST-297S > PST-287K > PST-WT. Corroboratively, circular dichroism analysis of PST peptides revealed significant differences in global structures (e.g. the order of propensity to form -helix was: PST-297S approximate to PST-287K > PST-WT). This study provides a molecular basis for enhanced potencies/efficacies of human PST variants (likely to occur in approximate to 300 million people worldwide) and has quantitative implications for inter-individual variations in glucose/insulin homeostasis

Catestatin Gly364Ser Variant Alters Systemic Blood Pressure and the Risk for Hypertension in Human Populations via Endothelial Nitric Oxide Pathway

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Gliomas induce and exploit microglial MT1-MMP expression for tumor expansion

Diffuse infiltration of glioma cells into normal brain tissue is considered to be a main reason for the unfavorable outcomes of patients with malignant gliomas. Invasion of glioma cells into the brain parenchyma is facilitated by metalloprotease-mediated degradation of the extracellular matrix. Metalloproteases are released as inactive pro-forms and get activated upon cleavage by membrane bound metalloproteases. Here, we show that membrane type 1 metalloprotease (MT1-MMP) is up-regulated in glioma-associated microglia, but not in the glioma cells. Overexpression of MT1-MMP is even lethal for glioma cells. Glioma-released factors trigger the expression and activity of MT1-MMP via microglial toll-like receptors and the p38 MAPK pathway, as deletion of the toll-like receptor adapter protein MyD88 or p38 inhibition prevented MT1-MMP expression and activity in cultured microglial cells. Microglial MT1-MMP in turn activates glioma-derived pro-MMP-2 and promotes glioma expansion, as shown in an ex vivo model using MT1-MMP-deficient brain tissue and a microglia depletion paradigm. Finally, MyD88 deficiency or microglia depletion largely attenuated glioma expansion in 2 independent in vivo models