Most nuclear systemic autoantigens are extremely disordered proteins: implications for the etiology of systemic autoimmunity

Patients with systemic autoimmune diseases usually produce high levels of antibodies to self-antigens (autoantigens). The repertoire of common autoantigens is remarkably limited, yet no readily understandable shared thread links these apparently diverse proteins. Using computer prediction algorithms, we have found that most nuclear systemic autoantigens are predicted to contain long regions of extreme structural disorder. Such disordered regions would generally make poor B cell epitopes and are predicted to be under-represented as potential T cell epitopes. Consideration of the potential role of protein disorder may give novel insights into the possible role of molecular mimicry in the pathogenesis of autoimmunity. The recognition of extreme autoantigen protein disorder has led us to an explicit model of epitope spreading that explains many of the paradoxical aspects of autoimmunity – in particular, the difficulty in identifying autoantigen-specific helper T cells that might collaborate with the B cells activated in systemic autoimmunity. The model also explains the experimentally observed breakdown of major histocompatibility complex (MHC) class specificity in peptides associated with the MHC II proteins of activated autoimmune B cells, and sheds light on the selection of particular T cell epitopes in autoimmunity. Finally, the model helps to rationalize the relative rarity of clinically significant autoimmunity despite the prevalence of low specificity/low avidity autoantibodies in normal individuals

Dynamics of allosteric regulation of the phospholipase C-γ isozymes upon recruitment to membranes

Numerous receptor tyrosine kinases and immune receptors activate phospholipase C-γ (PLC-γ) isozymes at membranes to control diverse cellular processes including phagocytosis, migration, proliferation, and differentiation. The molecular details of this process are not well understood. Using hydrogen-deuterium exchange mass spectrometry, we show that PLC-γ1 is relatively inert to lipid vesicles that contain its substrate, phosphatidylinositol 4,5-bisphosphate (PIP2), unless first bound to the kinase domain of the fibroblast growth factor receptor (FGFR1). Exchange occurs throughout PLC-γ1 and is exaggerated in PLC-γ1 containing an oncogenic substitution (D1165H) that allosterically activates the lipase. These data support a model whereby initial complex formation shifts the conformational equilibrium of PLC-γ1 to favor activation. This receptor-induced priming of PLC-γ1 also explains the capacity of a kinase-inactive fragment of FGFR1 to modestly enhance the lipase activity of PLC-γ1 operating on lipid vesicles but not a soluble analog of PIP2 and highlights potential cooperativity between receptor engagement and membrane proximity. Priming is expected to be greatly enhanced for receptors embedded in membranes and nearly universal for the myriad of receptors and co-receptors that bind the PLC-γ isozymes

Self-association of the Lentivirus protein, Nef

<p>Abstract</p> <p>Background</p> <p>The HIV-1 pathogenic factor, Nef, is a multifunctional protein present in the cytosol and on membranes of infected cells. It has been proposed that a spatial and temporal regulation of the conformation of Nef sequentially matches Nef's multiple functions to the process of virion production. Further, it has been suggested that dimerization is required for multiple Nef activities. A dimerization interface has been proposed based on intermolecular contacts between Nefs within hexagonal Nef/FynSH3 crystals. The proposed dimerization interface consists of the hydrophobic B-helix and flanking salt bridges between R105 and D123. Here, we test whether Nef self-association is mediated by this interface and address the overall significance of oligomerization.</p> <p>Results</p> <p>By co-immunoprecipitation assays, we demonstrated that HIV-1Nef exists as monomers and oligomers with about half of the Nef protomers oligomerized. Nef oligomers were found to be present in the cytosol and on membranes. Removal of the myristate did not enhance the oligomerization of soluble Nef. Also, SIVNef oligomerizes despite lacking a dimerization interface functionally homologous to that proposed for HIV-1Nef. Moreover, HIV-1Nef and SIVNef form hetero-oligomers demonstrating the existence of homologous oligomerization interfaces that are distinct from that previously proposed (R105-D123). Intracellular cross-linking by formaldehyde confirmed that SF2Nef dimers are present in intact cells, but surprisingly self-association was dependent on R105, but not D123. SIV_MAC239Nef can be cross-linked at its only cysteine, C55, and SF2Nef is also cross-linked, but at C206 instead of C55, suggesting that Nefs exhibit multiple dimeric structures. ClusPro dimerization analysis of HIV-1Nef homodimers and HIV-1Nef/SIVNef heterodimers identified a new potential dimerization interface, including a dibasic motif at R105-R106 and a six amino acid hydrophobic surface.</p> <p>Conclusions</p> <p>We have demonstrated significant levels of intracellular Nef oligomers by immunoprecipitation from cellular extracts. However, our results are contrary to the identification of salt bridges between R105 and D123 as necessary for self-association. Importantly, binding between HIV-1Nef and SIVNef demonstrates evolutionary conservation and therefore significant function(s) for oligomerization. Based on modeling studies of Nef self-association, we propose a new dimerization interface. Finally, our findings support a stochastic model of Nef function with a dispersed intracellular distribution of Nef oligomers.</p