Diversity of Structural, Dynamic, and Environmental Effects Explain a Distinctive Functional Role of Transmembrane Domains in the Insulin Receptor Subfamily.

Human InsR, IGF1R, and IRR receptor tyrosine kinases (RTK) of the insulin receptor subfamily play an important role in signaling pathways for a wide range of physiological processes and are directly associated with many pathologies, including neurodegenerative diseases. The disulfide-linked dimeric structure of these receptors is unique among RTKs. Sharing high sequence and structure homology, the receptors differ dramatically in their localization, expression, and functions. In this work, using high-resolution NMR spectroscopy supported by atomistic computer modeling, conformational variability of the transmembrane domains and their interactions with surrounding lipids were found to differ significantly between representatives of the subfamily. Therefore, we suggest that the heterogeneous and highly dynamic membrane environment should be taken into account in the observed diversity of the structural/dynamic organization and mechanisms of activation of InsR, IGF1R, and IRR receptors. This membrane-mediated control of receptor signaling offers an attractive prospect for the development of new targeted therapies for diseases associated with dysfunction of insulin subfamily receptors.

Amyloid Precursor Protein Changes Arrangement in a Membrane and Its Structure Depending on the Cholesterol Content.

One of the hallmarks of Alzheimer's disease (AD) is the accumulation of amyloid beta (Aß) peptides in the brain. The processing of amyloid precursor protein (APP) into Aß is dependent on the location of APP in the membrane, membrane lipid composition and, possibly, presence of lipid rafts. In this study, we used atomic force microscopy (AFM) to investigate the interaction between transmembrane fragment APP672-726 (corresponding to Aß1-55) and its amyloidogenic mutant L723P with membranes combining liquid-ordered and liquid-disordered lipid phases. Our results demonstrated that most of the APP672-726 is located either in the liquid-disordered phase or at the boundary between ordered and disordered phases, and hardly ever in rafts. We did not notice any major changes in the domain structure induced by APP672-726. In membranes without cholesterol APP672-726, and especially its amyloidogenic mutant L723P formed annular structures and clusters rising above the membrane. Presence of cholesterol led to the appearance of concave membrane regions up to 2 nm in depth that were deeper for wild type APP672-726. Thus, membrane cholesterol regulates changes in membrane structure and permeability induced by APP that might be connected with further formation of membrane pores.

Dimeric states of transmembrane domains of insulin and IGF-1R receptors: Structures and possible role in activation.

Despite the biological significance of insulin signaling, the molecular mechanisms of activation of the insulin receptor (IR) and other proteins from its family remain elusive. Current hypothesis on signal transduction suggests ligand-triggered structural changes in the extracellular domain followed by transmembrane (TM) domains closure and dimerization leading to trans-autophosphorylation and kinase activity in intracellular segments of the receptor. Using NMR spectroscopy, we detected dimerization of isolated TM segments of IR in different membrane-mimicking environments and observed multiple signals of NH groups of protein backbone possibly corresponding to several dimer conformations. Taking available experimental data as constraints, several atomistic models of dimeric TM domains of IR and insulin-like growth factor 1 (IGF-1R) receptors were elaborated. Molecular dynamics simulations of IR ectodomain revealed noticeable collective movements potentially responsible for closure of the C-termini of FnIII-3 domains and spatial approaching of TM helices upon insulin-induced receptor activation. In addition, we demonstrated that the intracellular part of the receptor does not impose restrictions on the positioning of TM helices in the membrane. Finally, we used two independent structure prediction methods to generate a series of dimer conformations followed by their cluster analysis and dimerization free energy estimation to select the best dimer models. Biological relevance of the later was further tested via comparison of the hydrophobic organization of TM helices for both wild-type receptors and their mutants. Based on these data, the ability of several segments from other proteins to functionally replace IR and/or IGF-1R TM domains was explained.

Transmembrane Peptides as a New Strategy to Inhibit Neuraminidase-1 Activation.

Sialidases, or neuraminidases, are involved in several human disorders such as neurodegenerative, infectious and cardiovascular diseases, and cancers. Accumulative data have shown that inhibition of neuraminidases, such as NEU1 sialidase, may be a promising pharmacological target, and selective inhibitors of NEU1 are therefore needed to better understand the biological functions of this sialidase. In the present study, we designed interfering peptides (IntPep) that target a transmembrane dimerization interface previously identified in human NEU1 that controls its membrane dimerization and sialidase activity. Two complementary strategies were used to deliver the IntPep into cells, either flanked to a TAT sequence or non-tagged for solubilization in detergent micelles. Combined with molecular dynamics simulations and heteronuclear nuclear magnetic resonance (NMR) studies in membrane-mimicking environments, our results show that these IntPep are able to interact with the dimerization interface of human NEU1, to disrupt membrane NEU1 dimerization and to strongly decrease its sialidase activity at the plasma membrane. In conclusion, we report here new selective inhibitors of human NEU1 of strong interest to elucidate the biological functions of this sialidase.