Conformational disorder in phosphopeptides: solution studies by CD and NMR techniques

In the last few years intrinsically disordered proteins (IDPs) have received great attention from the scientific community as they participate in several important biological processes and diseases. The intrinsic disorder and flexibility of IDPs grant them a number of advantages with respect to ordered proteins, such as conformational plasticity to bind several targets, a large interaction surface, involvement in high specificity/low affinity interactions, enhanced binding kinetics. It is assumed that post-translational modifications such as phosphorylation can stimulate structural rearrangement in IDPs and facilitate their binding to partners. To better understand at a structural level the multifaceted mechanisms that govern molecular recognition processes involving IDPs, we designed, synthesized by solid phase methods, and structurally characterized unstructured peptides. These molecules contain a putative disordered module, flanked at either the N- or C-terminal ends by a different phosphorylated amino acid (serine or threonine) to mimick the effects of phosphorylation. The absence of an ordered state in the designed peptides was proved experimentally by CD and NMR conformational studies that were carried out under different solution conditions

NMR STRUCTURAL AND BINDING STUDIES OF RECOMBINANT PROTEINS OF BIOTECHNOLOGICAL INTEREST

My PhD thesis has been focused on studies carried out by different experimental and computational techniques, such as NMR (Nuclear Magnetic Resonance), SPR (Surface Plasmon Resonance), ITC (Isothermal Titration Calorimetry), molecular docking, mutagenesis, of Sam (Sterile alpha motif) domains containing proteins that play important roles in physiological or pathological processes. Protein-protein interactions are essential for the assembly, regulation, and localization of functional protein complexes in the cell. The analysis of these interactions at molecular level is of great interest in many biotechnological fields, as it provides useful information for the design of molecules able to mimic the binding sites and thus, presenting potential therapeutic applications. Protein-protein associations are mediated by specific domains, such as Sam domains. Within this thesis two heterotypic Sam-Sam interactions were studied in details: 1) the EphA2-Sam/Odin-Sam1 complex; 2) the Odin-Sam1/Arap3-Sam association. The tyrosine kinase receptor EphA2 plays a fundamental role in tumorigenesis. The process of EphA2 endocytosis and consequent degradation has been investigated as potential route to reduce tumor malignancy. Odin belongs to the ANKS (Ankyrin repeat domain containing and Sam) protein family; it contains two Sam domains in tandem (Sam1 and Sam2), and is able to regulate EphA2 receptor endocytosis. Instead, Arap3 (Arf GAP, Rho GAP, Ankyrin repeat and PH domains) is a protein involved in the phosphoinositol-3-kinase (PI3K) signaling pathways, which regulates biological processes connected to cell motility. Firstly, by multidimensional (2D and 3D) NMR methods, the solution structure of Odin-Sam1 was determined. It consists of five -helices and represents a canonical Sam domain fold. Afterwards, binding studies with EphA2-Sam, and Arap3-Sam were conducted. SPR and ITC experiments revealed a low micromolar binding affinity of Odin-Sam1 for both EphA2-Sam and Arap3-Sam. The reciprocal interaction surfaces of these Sam domains were identified by NMR chemical shift perturbation experiments, and 3D models of the complexes were built by molecular docking techniques. These studies suggested that Odin-Sam1/EphA2-Sam and Odin-Sam1/Arap3-Sam complexes might adopt the canonical Sam-Sam interaction topology called "Mid-Loop/End-Helix", where the central portion of Odin-Sam1 and the C-terminal helix of either Arap3-Sam and EphA2-Sam contribute the binding surfaces. A peptide, named Sam3, which encompasses the central portion of the Odin-Sam1 involved in complexes formation with EphA2-Sam and Arap3-Sam, together with its C-terminal helix, was synthesized and analyzed by 2D NMR techniques. The peptide reveals unstructured in phosphate buffer, whereas it shows propensity to adopt more ordered helical structures in water/trifluoroethanol mixtures. Based on the new structural insights gained within my thesis, libraries of molecules (peptides and peptido-mimetics), that could selectively interfere with Odin-Sam1/EphA2-Sam or Odin-Sam1/Arap3-Sam associations, and prove useful in therapeutic and diagnostic applications, will be designed in the near future

Hunting for Novel Routes in Anticancer Drug Discovery: Peptides against Sam-Sam Interactions

Among the diverse protein binding modules, Sam (Sterile alpha motif) domains attract attention due to their versatility. They are present in different organisms and play many functions in physiological and pathological processes by binding multiple partners. The EphA2 receptor contains a Sam domain at the C-terminus (EphA2-Sam) that is able to engage protein regulators of receptor stability (including the lipid phosphatase Ship2 and the adaptor Odin). Ship2 and Odin are recruited by EphA2-Sam through heterotypic Sam-Sam interactions. Ship2 decreases EphA2 endocytosis and consequent degradation, producing chiefly pro-oncogenic outcomes in a cellular milieu. Odin, through its Sam domains, contributes to receptor stability by possibly interfering with ubiquitination. As EphA2 is upregulated in many types of tumors, peptide inhibitors of Sam-Sam interactions by hindering receptor stability could function as anticancer therapeutics. This review describes EphA2-Sam and its interactome from a structural and functional perspective. The diverse design strategies that have thus far been employed to obtain peptides targeting EphA2-mediated Sam-Sam interactions are summarized as well. The generated peptides represent good initial lead compounds, but surely many efforts need to be devoted in the close future to improve interaction affinities towards Sam domains and consequently validate their anticancer properties

Cancer-Related Mutations in the Sam Domains of EphA2 Receptor and Ship2 Lipid Phosphatase: A Computational Study

The lipid phosphatase Ship2 interacts with the EphA2 receptor by forming a heterotypic Sam (sterile alpha motif)–Sam complex. Ship2 works as a negative regulator of receptor endocytosis and consequent degradation, and anti-oncogenic effects in cancer cells should be induced by hindering its association with EphA2. Herein, a computational approach is presented to investigate the relationship between Ship2-Sam/EphA2-Sam interaction and cancer onset and further progression. A search was first conducted through the COSMIC (Catalogue of Somatic Mutations in Cancer) database to identify cancer-related missense mutations positioned inside or close to the EphA2–Sam and Ship2–Sam reciprocal binding interfaces. Next, potential differences in the chemical–physical properties of mutant and wild-type Sam domains were evaluated by bioinformatics tools based on analyses of primary sequences. Three-dimensional (3D) structural models of mutated EphA2–Sam and Ship2–Sam domains were built as well and deeply analysed with diverse computational instruments, including molecular dynamics, to classify potentially stabilizing and destabilizing mutations. In the end, the influence of mutations on the EphA2–Sam/Ship2–Sam interaction was studied through docking techniques. This in silico approach contributes to understanding, at the molecular level, the mutation/cancer relationship by predicting if amino acid substitutions could modulate EphA2 receptor endocytosis

Structure-Activity Relationship Investigations of Novel Constrained Chimeric Peptidomimetics of SOCS3 Protein Targeting JAK2

SOCS3 (suppressor of cytokine signaling 3) protein suppresses cytokine-induced inflammation and its deletion in neurons or immune cells increases the pathological growth of blood vessels. Recently, we designed several SOCS3 peptidomimetics by assuming as template structures the interfacing regions of the ternary complex constituted by SOCS3, JAK2 (Janus Kinase 2) and gp130 (glycoprotein 130) proteins. A chimeric peptide named KIRCONG chim, including non-contiguous regions demonstrated able to bind to JAK2 and anti-inflammatory and antioxidant properties in VSMCs (vascular smooth muscle cells). With the aim to improve drug-like features of KIRCONG, herein we reported novel cyclic analogues bearing different linkages. In detail, in two of them hydrocarbon cycles of different lengths were inserted at positions i/i+5 and i/i+7 to improve helical conformations of mimetics. Structural features of cyclic compounds were investigated by CD (Circular Dichroism) and NMR (Nuclear Magnetic Resonance) spectroscopies while their ability to bind to catalytic domain of JAK2 was assessed through MST (MicroScale Thermophoresis) assay as well as their stability in biological serum. Overall data indicate a crucial role exerted by the length and the position of the cycle within the chimeric structure and could pave the way to the miniaturization of SOCS3 protein for therapeutic aims