The Role of Intestinal Fatty Acid Binding Proteins in Protecting Cells from Fatty Acid Induced Impairment of Mitochondrial Dynamics and Apoptosis

Background/Aims: The conformation, folding and lipid binding properties of the intestinal fatty acid binding proteins (IFABP) have been extensively investigated. In contrast, the functional aspects of these proteins are not understood and matter of debates. In this study, we aim to address the deleterious effects of FA overload on cellular components, particularly mitochondria; and how IFABP helps in combating this stress by restoring the mitochondrial dynamics. Methods: In the present study the functional aspect of IFABP under conditions of lipid stress was studied by a string of extensive in-cell studies; flow cytometry by fluorescence-activated cell sorting (FACS), confocal imaging, western blotting and quantitative real time PCR. We deployed ectopic expression of IFABP in rescuing cells under the condition of lipid stress. Again in order to unveil the mechanistic insights of functional traits, we arrayed extensive computational approaches by means of studying centrality calculations along with protein-protein association and ligand induced cluster dissociation. While addressing its functional importance, we used FCS and in-silico computational analyses, to show the structural distribution and the underlying mechanism of IFABP’s action. Results: Ectopic expression of IFABP in HeLa cells has been found to rescue mitochondrial morphological dynamics and restore membrane potential, partially preventing apoptotic damage induced by the increased FAs. These findings have been further validated in the functionally relevant intestinal Caco-2 cells, where the native expression of IFABP protects mitochondrial morphology from abrogation induced by FA overload. However, this native level expression is insufficient to protect against apoptotic cell death, which is rescued, at least partially in cells overexpressing IFABP. In addition, shRNA mediated IFABP knockdown in Caco-2 cells compromises mitochondrial dynamics and switches on intrinsic apoptotic pathways under FA-induced metabolic stress. Conclusion: To summarize, the present study implicates functional significance of IFABP in controlling ligand-induced damage in mitochondrial dynamics and apoptosis

Specifically bound lambda repressor dimers promote adjacent non-specific binding

<div><p>Genetic switches frequently include DNA loops secured by proteins. Recent studies of the lambda bacteriophage repressor (CI), showed that this arrangement in which the protein links two sets of three operators separated by approximately 2.3 kbp, optimizes both the stability and dynamics of DNA loops, compared to an arrangement with just two sets of two operators. Because adjacent dimers interact pairwise, we hypothesized that the odd number of operators in each set of the lambda regulatory system might have evolved to allow for semi-specific, pair-wise interactions that add stability to the loop while maintaining it dynamic. More generally, additional CI dimers may bind non-specifically to flanking DNA sequences making the genetic switch more sensitive to CI concentration. Here, we tested this hypothesis using spectroscopic and imaging approaches to study the binding of the lambda repressor (CI) dimer protein to DNA fragments. For fragments with only one operator and a short flanking sequence, fluorescence correlation spectroscopy measurements clearly indicated the presence of two distinct DNA-CI complexes; one is thought to have a non-specifically bound CI dimer on the flanking sequence. Scanning force micrographs of CI bound to DNA with all six operators revealed wild-type or mutant proteins bound at operator positions. The number of bound, wild-type proteins increased with CI concentration and was larger than expected for strictly specific binding to operators. In contrast, a mutant that fails to oligomerize beyond a dimer, D197G, only bound to operators. These data are evidence that CI cooperativity promotes oligomerization that extends from operator sites to influence the thermodynamics and kinetics of CI-mediated looping.</p></div

Spatiotemporal Analysis of K‑Ras Plasma Membrane Interactions Reveals Multiple High Order Homo-oligomeric Complexes

Self-assembly of plasma membrane-associated Ras GTPases has major implications to the regulation of cell signaling. However, the structural basis of homo-oligomerization and the fractional distribution of oligomeric states remained undetermined. We have addressed these issues by deciphering the distribution of dimers and higher-order oligomers of K-Ras4B, the most frequently mutated Ras isoform in human cancers. We focused on the constitutively active G12V K-Ras and two of its variants, K101E and K101C/E107C, which respectively destabilize and stabilize oligomers. Using raster image correlation spectroscopy and number and brightness analysis combined with fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and electron microscopy in live cells, we show that G12V K-Ras exists as a mixture of monomers, dimers and larger oligomers, while the K101E mutant is predominantly monomeric and K101C/E107C is dominated by oligomers. This observation demonstrates the ability of K-Ras to exist in multiple oligomeric states whose population can be altered by interfacial mutations. Using molecular modeling and simulations we further show that K-Ras uses two partially overlapping interfaces to form compositionally and topologically diverse oligomers. Our results thus provide the first detailed insight into the multiplicity, structure, and membrane organization of K-Ras homomers

Specifically bound lambda repressor dimers promote adjacent non-specific binding - Fig 3

<p><b>Variations in the numbers of particles (<i>a</i>) and counts per particle (<i>b</i>) as a function of CI protein concentration.</b> Squares (filled) and circles (open) correspond to WT DNA constructs containing the OL3 or OR3 sites, respectively.</p

The CI concentrations at mid-points of the first and second transitions for titrations of CI with the indicated DNA constructs as shown in Figs 3 and 4.

<p>These are indicative measures of relative binding affinities for the different CI operators.</p

Regulation of bacteriophage gene expression by CI.

<p>(Upper) Tetramers, formed through lateral interactions of adjacent CI dimers (red circles) bound to high affinity “operator” (green) sites, inhibit transcription from the lytic promoters <i>pL</i> and <i>pR</i>. (Middle) The dimers bound at <i>OL</i>1 and <i>OL2</i> may interact head-to-head with those at <i>OR1</i> and <i>OR2</i>, to stabilize a DNA loop (dotted curve) to juxtapose and recruit dimers at the strong <i>OL3</i> and weaker OR3 sites which also interact head-to-head. The additional dimer on OR3 inhibits transcription from <i>pRM</i>. (Lower) The CI dimers at <i>OL3</i> and <i>OR3</i> are still available for lateral pairwise interaction and may cooperatively stabilize other dimers to adjacent, non-specific (gray) sites.</p

Number of CI dimer that bind during the first (<i>n</i>_<i>1</i>) and second (<i>n</i>_<i>2</i>) transitions.

<p>Number of CI dimer that bind during the first (<i>n</i>_<i>1</i>) and second (<i>n</i>_<i>2</i>) transitions.</p