ABSTRACT
We developed a highly sensitive silicon platform, suitable to assess the molecular organization of protein samples. Prototype platforms were obtained using different electrochemical protocols for the electrodeposition of Ag-nanoparticles onto the hydrogenated silicon surface. A platform with high Surface Enhanced Raman Scattering efficiency was selected based on the surface coverage and the number density of particles size distribution. The performance of the platform was determined by studying the interaction of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein with the substrate according to its molecular organization. The chemical and structural characteristics of MARCKS molecules were examined under two configurations: i) a disordered distribution given by a MARCKS solution drop deposited onto the platform and, ii) a compact monolayer transferred to the platform by the Langmuir-Blodgett method. Raman spectra show vibrational bands of Phenylalanine and Lysine residues specific for the protein effector domain, and evidence the presence of alpha helix structure in both configurations. Moreover, we distinguished the supramolecular order between the compact monolayer and random molecular distribution. The platforms containing Ag-nanoparticles are suitable for studies of protein structure and interactions, advancing a methodological strategy for our long term goal, which is to explore the interaction of proteins with model membranes.
Subject(s)
Myristoylated Alanine-Rich C Kinase Substrate/chemistry , Spectrum Analysis, Raman/methods , Electroplating , Humans , Metal Nanoparticles/chemistry , Protein Conformation, alpha-Helical , Protein Domains , Silicon/chemistry , Silver/chemistry , Surface PropertiesABSTRACT
Blood coagulation involves activation of platelets and coagulation factors. At the interface of these two processes resides the lipid phosphatidylserine. Activated platelets expose phosphatidylserine on their outer membrane leaflet and activated clotting factors assemble into enzymatically active complexes on the exposed lipid, ultimately leading to the formation of fibrin. Here, we describe how small peptide and peptidomimetic probes derived from the lipid binding domain of the protein myristoylated alanine-rich C-kinase substrate (MARCKS) bind to phosphatidylserine exposed on activated platelets and thereby inhibit fibrin formation. The MARCKS peptides antagonize the binding of factor Xa to phosphatidylserine and inhibit the enzymatic activity of prothrombinase. In whole blood under flow, the MARCKS peptides colocalize with, and inhibit fibrin cross-linking, of adherent platelets. In vivo, we find that the MARCKS peptides circulate to remote injuries and bind to activated platelets in the inner core of developing thrombi.
Subject(s)
Blood Coagulation Factors/metabolism , Multiprotein Complexes/metabolism , Myristoylated Alanine-Rich C Kinase Substrate/metabolism , Peptides/metabolism , Phosphatidylserines/metabolism , Blood Coagulation Factors/chemistry , Blood Platelets/metabolism , Factor Xa/chemistry , Factor Xa/metabolism , Fibrin/chemistry , Fibrin/metabolism , Humans , Liposomes , Multiprotein Complexes/chemistry , Myristoylated Alanine-Rich C Kinase Substrate/chemistry , Peptides/chemistry , Peptides/pharmacology , Phosphatidylserines/chemistry , Platelet Activation , Protein Binding/drug effects , Proteolysis , Surface Plasmon Resonance , Thromboplastin/metabolismABSTRACT
Alzheimer's disease (AD) is the most common neurodegenerative disease, but it remains an intractable condition. Its pathogenesis is predominantly attributed to the aggregation and transmission of two molecules, Aß and tau; however, other pathological mechanisms are possible. Here, we reveal that phosphorylation of MARCKS, a submembrane protein that regulates the stability of the actin network, occurs at Ser46 prior to aggregation of Aß and is sustained throughout the course of AD in human and mouse brains. Furthermore, HMGB1 released from necrotic or hyperexcitatory neurons binds to TLR4, triggers the specific phosphorylation of MARCKS via MAP kinases, and induces neurite degeneration, the classical hallmark of AD pathology. Subcutaneous injection of a newly developed monoclonal antibody against HMGB1 strongly inhibits neurite degeneration even in the presence of Aß plaques and completely recovers cognitive impairment in a mouse model. HMGB1 and Aß mutually affect polymerization of the other molecule, and the therapeutic effects of the anti-HMGB1 monoclonal antibody are mediated by Aß-dependent and Aß-independent mechanisms. We propose that HMGB1 is a critical pathogenic molecule promoting AD pathology in parallel with Aß and tau and a new key molecular target of preclinical antibody therapy to delay the onset of AD.
