Molecular Modeling, Interacting Residues and other Structural Analyses for Human SOCS3, Gp130 and JAK Proteins: A Detailed Computational Approach for Proteins Involved in Feedback Inhibition

Background: When STAT3 is activated only by the IL6 family of proteins, then gp130 (having a phosphopeptide motif) interacts with human SOCS3 which further binds to JAK and inhibits its protein kinase activity. Interaction of gp130 with SOCS3 targets only the IL-6 signaling cascade. The interaction occurs when SOCS3 binds to a particular motif on gp130 (centered upon pTyr759) after its phosphorylation. Previously, wet laboratory studies were done but computational exploration for the participating residues remained unexplored. Methodology: The 3D structure of human SOCS3 protein was modeled and its stereo-chemical parameters were satisfied. Crystallographic structures of gp130-phosphopeptide and JAK were studied. After protein docking, the complex underwent minimization and molecular dynamics simulation. Different stability parameters and binding patterns with residues were evaluated Results, Discussion and Conclusion: The best modeled structure of SOCS3 protein was selected and found that it had three helices and seven sheets interspersed with coils. Arg133, Tyr137 and Tyr98 from SOCS3 formed manifold binding patterns with gp130 (mainly with pTyr759 and Glu758). Lys62, Lys63 and Arg65 from SOCS3 were also found to interact with Val762 of gp130. Interactions with JAK were also studied. Residue 53, 62-65, 98, 133, 136 and 137 formed the predominant binding pockets in SOCS3. They can serve as important target sites as well. Altogether, it created elctrostatically charged pockets to accommodate the partner proteins for each other. Gp130 phosphopeptide was observed to be tightly accommodated in the electrostatically positive zones on SOCS3 surface. Net area for solvent accessibility was also found to get drastically reduced implying high participation of residues. Earlier studies documented that the interaction of these three proteins occurs with affinity and have satisfactory association with each other. Here in this study, free energy of binding for the triple protein interaction through the ΔG values helped to infer that SOCS3 interacted spontaneously (in thermodynamic sense). Many helical conformations formed coiled-coils providing high flexibility to interact spontaneously. Most of the interactions were through the responsible SH2 domain (46-127 residue length) of SOCS3. Residues 53, 62-64 and 98 formed coils while the residue number 137adopted sheet conformation from coils. Future Scope: This study shall instigate to block the gp130-binding sites of SOCS3 through targeting of drugs, thereby preventing SOCS3-gp130 interaction. This would allow JAK-STAT signaling cascade which is paramount for several biological functions

Experimental Characterization of the Interaction between the N-Terminal SH3 Domain of Crkl and C3G

Crkl is a protein involved in the onset of several cancer pathologies that exerts its function only through its protein–protein interaction domains, a SH2 domain and two SH3 domains. SH3 domains are small protein interaction modules that mediate the binding and recognition of proline-rich sequences. One of the main physiological interactors of Crkl is C3G (also known as RAPGEF1), an interaction with key implications in regulating cellular growth and differentiation, cell morphogenesis and adhesion processes. Thus, understanding the interaction between Crkl and C3G is fundamental to gaining information about the molecular determinants of the several cancer pathologies in which these proteins are involved. In this paper, through a combination of fast kinetics at different experimental conditions and site-directed mutagenesis, we characterize the binding reaction between the N-SH3 domain of Crkl and a peptide mimicking a specific portion of C3G. Our results show a clear effect of pH on the stability of the complex, due to the protonation of negatively charged residues in the binding pocket of N-SH3. Our results are discussed under the light of previous work on SH3 domains.

Discovery of an exosite on the SOCS2-SH2 domain that enhances SH2 binding to phosphorylated ligands

Suppressor of cytokine signaling (SOCS)2 protein is a key negative regulator of the growth hormone (GH) and Janus kinase (JAK)-Signal Transducers and Activators of Transcription (STAT) signaling cascade. The central SOCS2-Src homology 2 (SH2) domain is characteristic of the SOCS family proteins and is an important module that facilitates recognition of targets bearing phosphorylated tyrosine (pTyr) residues. Here we identify an exosite on the SOCS2-SH2 domain which, when bound to a non-phosphorylated peptide (F3), enhances SH2 affinity for canonical phosphorylated ligands. Solution of the SOCS2/F3 crystal structure reveals F3 as an α-helix which binds on the opposite side of the SH2 domain to the phosphopeptide binding site. F3:exosite binding appears to stabilise the SOCS2-SH2 domain, resulting in slower dissociation of phosphorylated ligands and consequently, enhances binding affinity. This biophysical enhancement of SH2:pTyr binding affinity translates to increase SOCS2 inhibition of GH signaling.

Molecular associations, clinical, and prognostic implications of PTPN11 mutations in acute myeloid leukemia (Alliance)

Prognostic factors associated with chemotherapy outcomes in patients with acute myeloid leukemia (AML) are extensively reported, and one gene whose mutation is recognized as conferring resistance to several newer targeted therapies is protein tyrosine phosphatase non-receptor type 11 (PTPN11). The broader clinical implications of PTPN11 mutations in AML are still not well understood. The objective of this study was to determine which cytogenetic abnormalities and gene mutations co-occur with PTPN11 mutations and how PTPN11 mutations impact outcomes of patients treated with intensive chemotherapy. We studied 1,725 newly diagnosed AML patients (excluding acute promyelocytic leukemia) enrolled onto the Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology trials. In 140 PTPN11-mutated patient samples, PTPN11 most commonly co-occurred with mutations in NPM1, DNMT3A, and TET2. PTPN11 mutations were relatively common in patients with an inv(3)(q21q26)/t(3;3)(q21;q26) and a normal karyotype but were very rare in patients with typical complex karyotype and core-binding factor AML. Mutations in the N-terminal SH2 domain of PTPN11 were associated with a higher early death rate than those in the phosphatase domain. PTPN11 mutations did not affect outcomes of NPM1-mutated patients, but these patients were less likely to have co-occurring kinase mutations (i.e., FLT3-ITD), suggesting activation of overlapping signaling pathways. However, in AML patients with wild-type NPM1, PTPN11 mutations were associated with adverse patient outcomes providing a rationale to study the biology and treatment approaches in this molecular group.

miR-514a-3p: a novel SHP-2 regulatory miRNA that modulates human cytotrophoblast proliferation

Src homology-2 (SH2) domain containing protein tyrosine phosphatase 2 (SHP-2), encoded by the PTPN11 gene, forms a central component of multiple signalling pathways and is required for insulin-like growth factor (IGF) induced placental growth. Altered expression of SHP-2 is associated with aberrant placental and fetal growth indicating that drugs modulating SHP-2 expression may improve adverse pregnancy outcome associated with altered placental growth. Placental PTPN11/SHP-2 expression is controlled by microRNAs (miRNAs) so SHP-2 regulatory miRNAs may have therapeutic potential, however the individual miRNA(s) regulating placental SHP-2 expression remain to be established. We performed in-silico analysis of 3’UTR target prediction databases to identify libraries of HeLa cells transfected with individual miRNA-mimetics, enriched in potential SHP-2 regulatory miRNAs. Analysis of PTPN11 levels by qPCR revealed that miR-758-3p increased, whilst miR-514a-3p reduced PTPN11 expression. miR-514a-3p and miR-758-3p expression within the human placenta was confirmed by qPCR; miR-514a-3p (but not miR-758-3p) levels inversely correlated with PTPN11 expression. To assess the interaction between these miRNAs and PTPN11/SHP-2, specific mimetics were transfected into first trimester human placental explants and explants cultured for up to 4 days. Overexpression of miR-514a-3p, but not miR-758-3p, significantly reduced PTPN11 and SHP-2 expression. microRNA-ribonucleoprotein complex (miRNP)-associated mRNA assays confirmed that this interaction was direct. miR-514a-3p overexpression attenuated IGF-I induced trophoblast proliferation (BrdU incorporation). miR-758-3p did not alter trophoblast proliferation. These data demonstrate that by modulating SHP-2 expression, miR-514a-3p is a novel regulator of IGF- signalling and proliferation in the human placenta and may have therapeutic potential in pregnancies complicated by altered placental growth.

A novel approach of recombinant laterosporulin production using the N-SH2 domain of SHP-2

Background The current study was aimed at evaluating the role of the N-SH2 domain of SHP-2 as a partner protein in the expression of a toxic peptide, laterosporulin (LTS). We also investigated its effects on the formation of the disulfide bond and functional folding of the peptide in vitro. The N-SH2-LTS protein was expressed as a His-tagged fusion protein, capable of undergoing enzymatic cleavage. Results Based on the data presented herein, the total yield of the folded fusion protein from inclusion bodies was found to be about 105 mg/l, demonstrating a high-level of heterologous expression. After enzymatic cleavage, 1.5 mg of the folded recombinant laterosporulin was obtained from each 10 mg of the fusion protein. The purity of the recombinant laterosporulin was analyzed by RP-HPLC, to yield peptides with suitable purity (85%). Conclusions Our findings indicated the advantages of using the N-SH2 domain of SHP-2 as a rapid and easy approach not only in producing easy target proteins but also in its function as a chaperone. N-SH2 domain of SHP-2 can influence on the purification of laterosporulin at reasonable yield and in a cost-effective fashion. The N-SH2 domain of SHP-2 as a protein chaperone may be potentially favorable to produce other proteins with disulfide bonds.

Structural and functional analysis of target recognition by the lymphocyte adaptor protein LNK

The SH2B family of adaptor proteins, SH2-B, APS, and LNK are key modulators of cellular signalling pathways. Whilst SH2-B and APS have been partially structurally and biochemically characterised, to date there has been no such characterisation of LNK. Here we present two crystal structures of the LNK substrate recognition domain, the SH2 domain, bound to phosphorylated motifs from JAK2 and EPOR, and biochemically define the basis for target recognition. The LNK SH2 domain adopts a canonical SH2 domain fold with an additional N-terminal helix. Targeted analysis of binding to phosphosites in signalling pathways indicated that specificity is conferred by amino acids one- and three-residues downstream of the phosphotyrosine. Several mutations in LNK showed impaired target binding in vitro and a reduced ability to inhibit signalling, allowing an understanding of the molecular basis of LNK dysfunction in variants identified in patients with myeloproliferative disease.

Development of polymeric nanoparticles loaded with STAT3 inhibitory, Stattic, for targeted cancer therapy

Background: STAT3 is an oncogenic signaling pathway found constitutively active in many types of human malignancies and plays a key role in cancer progression. Stattic is a small molecule, which selectively inhibits SH2 domain of STAT3. In most of the studies, stattic has been proposed as a promising strategy for inhibition of STAT3 in cancer cells harboring constitutively active STAT3. However, lack of proper formulation due to the poor water solubility and low bioavailability of stattic is a major limitation for its usage in clinic. The aim of this project was to develop poly(ethylene glycole)-block-poly(caprolactone) (PEG-b-PCL)-based polymeric micelles loaded with stattic and evaluate drug encapsulation efficiency and release in the developed formulations. Methods: In this experimental study, to prepare stattic loaded micellar formulations, co-solvent evaporation method was used. Mean diameter and polydispersity index (PDI) of micelles were defined by light scattering method. Encapsulated drug levels were measured using high performance liquid chromatography (HPLC). Data were analyzed using Graph pad prism software through one-way ANOVA analysis of variance. Results: Stattic was loaded in the polymeric micelles with encapsulation efficiency ranging from 40 to 73%. Drug loaded micelles were measured between 90 to 130 nm in size. PDI was obtained 0.3-1 and encapsulation of stattic in Polyethylene glycol-block-poly(α-benzyl carboxylate ε-caprolactone(PEG-b-PBCL) micellar formulation resulted in more than 6-fold increase in the water solubility of stattic (0.36 vs. 0.06 mg/mL). Respecting to high encapsulation efficiency, two micellar formulations were selected for further analysis that both of them released 70-80% of drug within the first hour, indicated burst release of drug. Conclusion: These findings show that PEG-b-PBCL copolymers can be a suitable vehicle for solubilization of stattic.