SH3 Domain Research Articles

Peptide-binding specificity prediction using fine-tuned protein structure prediction networks

Peptide-binding proteins play key roles in biology, and predicting their binding specificity is a long-standing challenge. While considerable protein structural information is available, the most successful current methods use sequence information alone, in part because it has been a challenge to model the subtle structural changes accompanying sequence substitutions. Protein structure prediction networks such as AlphaFold model sequence-structure relationships very accurately, and we reasoned that if it were possible to specifically train such networks on binding data, more generalizable models could be created. We show that placing a classifier on top of the AlphaFold network and fine-tuning the combined network parameters for both classification and structure prediction accuracy leads to a model with strong generalizable performance on a wide range of Class I and Class II peptide-MHC interactions that approaches the overall performance of the state-of-the-art NetMHCpan sequence-based method. The peptide-MHC optimized model shows excellent performance in distinguishing binding and non-binding peptides to SH3 and PDZ domains. This ability to generalize well beyond the training set far exceeds that of sequence-only models and should be particularly powerful for systems where less experimental data are available.

Dual inhibition of MYC and SLC39A10 by a novel natural product STAT3 inhibitor derived from Chaetomium globosum suppresses tumor growth and metastasis in gastric cancer

Gastric cancer remains one of the most common deadly diseases and lacks effective targeted therapies. In the present study, we confirmed that the signal transducer and activator of transcription 3 (STAT3) is highly expressed and associated with a poor prognosis in gastric cancer. We further identified a novel natural product inhibitor of STAT3, termed XYA-2, which interacts specifically with the SH2 domain of STAT3 (Kd= 3.29 μM) and inhibits IL-6-induced STAT3 phosphorylation at Tyr705 and nuclear translocation. XYA-2 inhibited the viability of seven human gastric cancer cell lines with 72-h IC50 values ranging from 0.5 to 0.7 μΜ. XYA-2 at 1 μΜ inhibited the colony formation and migration ability of MGC803 (72.6% and 67.6%, respectively) and MKN28 (78.5% and 96.6%, respectively) cells. In the in vivo studies, intraperitoneal administration of XYA-2 (10 mg/kg/day, 7 days/week) significantly suppressed 59.8% and 88.8% tumor growth in the MKN28-derived xenograft mouse model and MGC803-derived orthotopic mouse model, respectively. Similar results were obtained in a patient-derived xenograft (PDX) mouse model. Moreover, XYA-2 treatment extended the survival of mice bearing PDX tumors. The molecular mechanism studies based on transcriptomics and proteomics analyses indicated that XYA-2 might exert its anticancer activity by synergistically inhibiting the expression of MYC and SLC39A10, two downstream genes of STAT3 in vitro and in vivo. Together, these findings suggested that XYA-2 may be a potent STAT3 inhibitor for treating gastric cancer, and dual inhibition of MYC and SLC39A10 may be an effective therapeutic strategy for STAT3-activated cancer.

Monobody Inhibitor Selective to the Phosphatase Domain of SHP2 and its Use as a Probe for Quantifying SHP2 Allosteric Regulation

SHP2 is a phosphatase/adaptor protein that plays an important role in various signaling pathways. Its mutations are associated with cancers and developmental diseases. SHP2 contains a protein tyrosine phosphatase (PTP) and two SH2 domains. Selective inhibition of these domains has been challenging due to the multitude of homologous proteins in the proteome. Here, we developed a monobody, synthetic binding protein, that bound to and inhibited the SHP2 PTP domain. It was selective to SHP2 PTP over close homologs. A crystal structure of the monobody-PTP complex revealed that the monobody bound both highly conserved residues in the active site and less conserved residues in the periphery, rationalizing its high selectivity. Its epitope overlapped with the interface between the PTP and N-terminal SH2 domains that is formed in auto-inhibited SHP2. By using the monobody as a probe for the accessibility of the PTP active site, we developed a simple, nonenzymatic assay for the allosteric regulation of SHP2. The assay showed that, in the absence of an activating phospho-Tyr ligand, wild-type SHP2 and the “PTP-dead” C459E mutant were predominantly in the closed state in which the PTP active site is inaccessible, whereas the E76K and C459S mutants were in the open, active state. It also revealed that previously developed monobodies to the SH2 domains, ligands lacking a phospho-Tyr, weakly favored the open state. These results provide corroboration for a conformational equilibrium underlying allosteric regulation of SHP2, provide powerful tools for characterizing and controlling SHP2 functions, and inform drug discovery against SHP2.

The influence of pH on the structure and stability of the Grb2 dimer reveals changes in the inter-domain and molecular interaction: Could it be a modulation mechanism?

Cancer cells present an increased replicative potential as a hallmark. The increased replication leads to a higher intracellular pH. Grb2, an adapter protein, is mainly involved in several types of cancers due to its role in signaling pathways responsible for cell growth and proliferation. At pH 7, we observed a more compact structure, as seen by DLS and 1H NMR relaxation experiments, with high cooperativity within domains. On the other hand, we observed an increase in disordered structures at pH 8, with relative independence between domains characterized by higher melting temperatures and enthalpy of unfolding. CD and DLS corroborate with these observations at pH 8, conferring more flexibility among the domains, followed by lower unfolding cooperativity and increased hydrodynamic diameter at higher pH. In addition, 15N-HSQC chemical shift perturbations experiments showed significant differences in the positions of several amino acids spread on the Grb2 structure when pH was changed, which agrees with the previous results. Finally, the molecular dynamic analysis demonstrates that Grb2 presents a movement pattern where both SH3 domains move toward the center of the protein at pH 7. On the contrary, the pattern changes its direction at pH 8, where domains move outside the center of the protein, conferring a more elongated structure at higher pH. So, Grb2 presents significant structural and dynamic changes modulated by pH. If considering the role of Grb2 in cell signaling upstream, these conformational changes could be a critical mechanistic behavior of this protein, preventing/disrupting the stability of the cell signaling pathways related to cancer.

Site-Specific Phosphorylation of RTK KIT Kinase Insert Domain: Interactome Landscape Perspectives

The kinase insert domain (KID) of RTK KIT is a key recruitment region for downstream signalling proteins (DSPs). KID, as a multisite phosphorylation region, provides alternative recognition sites for DSPs and activates them by binding a phosphotyrosine (pY) to their SH2 domains. Significant steric, biochemical, and biophysical requirements must be fulfilled by each pair of interacting proteins as the adaptation of their configurations is mandatory for the selective activation of DSPs. The accurate 3D atomistic models obtained by modelling and molecular dynamics (MD) simulations of phosphorylated KID (p-KID) have been delivered to describe KID INTERACTOME. By taking phosphorylated KIDpY721 and the N-terminal SH2 domain of phosphatidylinositol 3-kinase (PI3K), a physiological partner of KID, we showed the two proteins are intrinsically disordered. Using 3D models of both proteins, we probe alternative orientations of KIDpY721 relative to the SH2 binding pocket using automatic docking (HADDOCK) and intuitive user-guided docking. This modelling yields to two possible models of the functionally related non-covalent complex KIDpY721/SH2, where one can be regarded as the first precursor to probe PI3K activation via KIT KID. We suggest that such generation of a KID/SH2 complex is best suited for future studies of the post-transduction effects of RTK KIT.

Neutron Reflectometry and Molecular Simulations Demonstrate HIV-1 Nef Homodimer Formation on Model Lipid Bilayers

The HIV-1 Nef protein plays a critical role in viral infectivity, high-titer replication in vivo, and immune escape of HIV-infected cells. Nef lacks intrinsic biochemical activity, functioning instead through interactions with diverse host cell signaling proteins and intracellular trafficking pathways. Previous studies have established an essential role for Nef homodimer formation at the plasma membrane for most if not all its functions. Here we combined neutron reflectometry of full-length myristoylated Nef bound to model lipid bilayers with molecular simulations based on previous X-ray crystal structures of Nef homodimers. This integrated approach provides direct evidence that Nef associates with the membrane as a homodimer with its structured core region displaced from the membrane for partner protein engagement. Parallel studies of a dimerization-defective mutant, Nef-L112D, demonstrate that the helical dimerization interface present in previous crystal structures stabilizes the membrane-bound dimer. X-ray crystallography of the Nef-L112D mutant in complex with the SH3 domain of the Nef-associated host cell kinase Hck revealed a monomeric 1:1 complex instead of the 2:2 dimer complex formed with wild-type Nef. Importantly, the crystal structure of the Nef-L112D core and SH3 interface are virtually identical to the wild-type complex, indicating that this mutation does not affect the overall Nef fold. These findings support the intrinsic capacity of Nef to homodimerize at lipid bilayers using structural features present in X-ray crystal structures of dimeric complexes.

Targeting Shank3 deficiency and paresthesia in autism spectrum disorder: A brief review.

Autism spectrum disorder (ASD) includes a group of multifactorial neurodevelopmental disorders characterized by impaired social communication, social interaction, and repetitive behaviors. Several studies have shown an association between cases of ASD and mutations in the genes of SH3 and multiple ankyrin repeat domain protein 3 (SHANK3). These genes encode many cell adhesion molecules, scaffold proteins, and proteins involved in synaptic transcription, protein synthesis, and degradation. They have a profound impact on all aspects of synaptic transmission and plasticity, including synapse formation and degeneration, suggesting that the pathogenesis of ASD may be partially attributable to synaptic dysfunction. In this review, we summarize the mechanism of synapses related to Shank3 in ASD. We also discuss the molecular, cellular, and functional studies of experimental models of ASD and current autism treatment methods targeting related proteins.

Src heterodimerically activates Lyn or Fyn to serve as targets for the diagnosis and treatment of esophageal squamous cell carcinoma.

Although Src is one of the oldest and most investigated oncoproteins, its function in tumor malignancy remains to be defined further. In this study, we demonstrated that the inhibition of Src activity by ponatinib effectively suppressed several malignant phenotypes of esophageal squamous cell carcinoma (ESCC) both in vitro and in vivo, whereas it did not produce growth-inhibitory effects on normal esophageal epithelial cells (NEECs). Importantly, we combined phosphoproteomics and several cellular and molecular biologic strategies to identify that Src interacted with the members of Src-family kinases (SFKs), such as Fyn or Lyn, to form heterodimers. Src interactions with Fyn and Lyn phosphorylated the tyrosine sites in SH2 (Fyn Tyr185 or Lyn Tyr183) and kinase domains (Fyn Tyr420 or Lyn Tyr397), which critically contributed to ESCC development. By contrast, Src could not form heterodimers with Fyn or Lyn in NEECs. We used RNA sequencing to comprehensively demonstrate that the inhibition of Src activity effectively blocked several critical tumor-promoting pathways, such as JAK/STAT, mTOR, stemness-related, and metabolism-related pathways. Results of the real-time polymerase chain reaction (RT-PCR) assay confirmed that Lyn and Fyn were critical effectors for the Src-mediated expression of tumor growth or metastasis-related molecules. Furthermore, results of the clinical ESCC samples showed that the hyperactivation of pSrc Tyr419, Fyn Tyr185 or Tyr420, and Lyn Tyr183 or Tyr397 could be biomarkers of ESCC prognosis. This study illustrates that Src/Fyn and Src/Lyn heterodimers serve as targets for the treatment of ESCC.

Viral Hemorrhagic Septicemia Virus Activates Integrated Stress Response Pathway and Induces Stress Granules to Regulate Virus Replication.

Virus infection activates integrated stress response (ISR) and stress granule (SG) formation and viruses counteract by interfering with SG assembly, suggesting an important role in antiviral defense. The infection of fish cells by Viral Hemorrhagic Septicemia Virus (VHSV), activates the innate immune recognition pathway and the production of type I interferon (IFN). However, the mechanisms by which VHSV interacts with ISR pathway regulating SG formation is poorly understood. Here, we demonstrate that fish cells respond to heat shock, oxidative stress and VHSV infection by forming SG that localized key SG marker, Ras GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1). We show that PKR-like endoplasmic reticulum kinase (PERK), but not (dsRNA)-dependent protein kinase (PKR), is required for VHSV-induced SG formation. Furthermore, in VHSV Ia infected cells, PERK activity is required for IFN production, antiviral signaling and viral replication. SG formation required active virus replication as individual VHSV Ia proteins or inactive virus did not induce SG. Cells lacking G3BP1 produced increased IFN, antiviral genes and viral mRNA, however viral protein synthesis and viral titers were reduced. We show a critical role of the activation of ISR pathway and SG formation highlighting a novel role of G3BP1 in regulating VHSV protein translation and replication.

Discovery of a Potent and Selective STAT5 PROTAC Degrader with Strong Antitumor Activity In Vivo in Acute Myeloid Leukemia.

STAT5 is an attractive therapeutic target for human cancers. We report herein the discovery of a potent and selective STAT5 degrader with strong antitumor activity in vivo. We first obtained small-molecule ligands with sub-micromolar to low micromolar binding affinities to STAT5 and STAT6 SH2 domains and determined co-crystal structures of three such ligands in complex with STAT5A. We successfully transformed these ligands into potent and selective STAT5 degraders using the PROTAC technology with AK-2292 as the best compound. AK-2292 effectively induces degradation of STAT5A, STAT5B, and phosphorylated STAT5 proteins in a concentration- and time-dependent manner in acute myeloid leukemia (AML) cell lines and demonstrates excellent degradation selectivity for STAT5 over all other STAT members. It exerts potent and specific cell growth inhibitory activity in AML cell lines with high levels of phosphorylated STAT5. AK-2292 effectively reduces STAT5 protein in vivo and achieves strong antitumor activity in mice at well-tolerated dose schedules.

IQGAP1 Is a Phosphotyrosine-Regulated Scaffold for SH2-Containing Proteins.

The scaffold protein IQGAP1 associates with over 150 interactors to influence multiple biological processes. The molecular mechanisms that underly spatial and temporal regulation of these interactions, which are crucial for proper cell functions, remain poorly understood. The receptor tyrosine kinase MET phosphorylates IQGAP1 on Tyr1510. Separately, Src homology 2 (SH2) domains mediate protein-protein interactions by binding specific phosphotyrosine residues. Here, we investigate whether MET-catalyzed phosphorylation of Tyr1510 of IQGAP1 regulates the docking of SH2-containing proteins. Using a peptide array, we identified SH2 domains from several proteins, including the non-receptor tyrosine kinases Abl1 and Abl2, that bind to the Tyr1510 of IQGAP1 in a phosphorylation-dependent manner. Using pure proteins, we validated that full-length Abl1 and Abl2 bind directly to phosphorylated Tyr1510 of IQGAP1. In cells, MET inhibition decreases endogenous IQGAP1 phosphorylation and interaction with endogenous Abl1 and Abl2, indicating that binding is regulated by MET-catalyzed phosphorylation of IQGAP1. Functionally, IQGAP1 modulates basal and HGF-stimulated Abl signaling. Moreover, IQGAP1 binds directly to MET, inhibiting its activation and signaling. Collectively, our study demonstrates that IQGAP1 is a phosphotyrosine-regulated scaffold for SH2-containing proteins, thereby uncovering a previously unidentified mechanism by which IQGAP1 coordinates intracellular signaling.

Effects of a key negatively charged residue mutation on the binding pathway of SH3 domain complex and ArkA peptide.

SH3 domains are common protein interaction domains found across all forms of life and serve important functions in cell signaling and cytoskeletal regulation. SH3 domains often bind to intrinsically disordered peptides (IDPs) which are peptides that lack a well-defined secondary structure. However, little is known about the binding mechanism in these complexes. Prior to this experiment, our lab has simulated the disordered ArkA peptide binding to the yeast SH3 domain AbpSH3, and it was observed that the binding proceeds through a flexible disordered encounter complex before reaching a fully bound state. While the encounter complex forms quickly, the slow step of binding is the transition from the disordered encounter ensemble to fully engaged state. We mutated glutamic acid to glutamine due to its interaction with the ArkA lysine (−3) in the bound state, a residue that is located at the center of 12-residue ArkA peptide and is required for binding. In this experiment, we used molecular dynamics simulations to simulate the binding between AbpSH3, which has a net negative charge, and ArkA, which has a net positive charge. We analyzed the effect a single residue mutation has on the transitioning of the ArkA peptide and AbpSH3 domain from the unbound stage to the disordered encounter complex and finally to the fully bound complex. We found that the mutated domain forms similar contacts with ArkA in both the encounter complex and bound state compared to the wild type, despite the disruption of the favorable interaction between glutamic acid and lysine. For future research, we will examine the effects of mutating the ArkA peptide on both the encounter complex and the fully bound state.

Charactering ionizable residue networks at SH2 interfaces in pH-sensitive proteins.

Intracellular pH (pHi) dynamics (7.0 to 7.8) regulate myriad cell processes but the molecular mechanisms driving these behaviors are largely unknown. While some pH-sensitive proteins use titratable histidine residues to sense physiological changes in pHi, others have unknown or histidine-independent mechanisms. Charged amino acids (Asp, Glu, and Lys) can have pKas that are up- or down-shifted into the physiological range depending on protein environment, but the role for these residues in regulating pH-dependent protein function is understudied. Here, we use computational and biochemical approaches to investigate pH sensors that use networks of cooperative ionizable residues with a cumulative physiological pKa. We performed computational analyses of two pH sensitive signaling proteins with unknown mechanisms: (phosphatidylinositol-3-kinase (PI3K) and SHP-2 tyrosine-protein phosphatase). In both proteins, we identified networks of ionizable residues with upshifted pKas at the inhibitory SH2 domain interface. We next used biochemical mutational analyses to probe the accuracy of our computational approaches by measuring pH sensitive activity of wild-type and mutant proteins in vitro. Importantly, because SH2 domains are structurally conserved, this pH-sensing mechanism could be conserved in a subset of kinases and phosphatases with inhibitory SH2 domains. We tested this hypothesis by identifying a network of ionizable residues in other signaling proteins that contain inhibitory SH2 domains. We analyzed JAK2 kinase, which has not been shown to have pH sensitive function, and identified an ionizable network at the SH2 domain interface. Future work will validate this JACK2 result biochemically and explore whether our computational approaches can be applied to identify pH sensitive proteins across the human proteome. Improved understanding of how ionizable networks respond to pH could reveal paths to better therapeutic targeting of these proteins in diseases with dysregulated pHi.

SH3 binding pathway and kinetics in the presence of salt.

Protein-protein interactions are involved in a wide range of cellular processes, like cell signaling, in which these interactions often bind to intrinsically disordered proteins. SH3 domains are common protein interaction domains which are typically negatively charged and typically bind proline-rich disordered segments, which are positively charged. This project focuses on the role of electrostatics in the binding pathway of the yeast protein Abp1 SH3 domain (AbpSH3) and the intrinsically disordered peptide ArkA. We are investigating the binding pathway by simulating ArkA-AbpSH3 binding using molecular dynamics in the presence of sodium chloride, which serves to screen electrostatic interactions. Previous simulations in the absence of salt showed that ArkA initially forms a disordered encounter complex with AbpSH3 before reaching the fully bound state. In the absence of salt, the encounter complex, characterized by many transient non-specific electrostatic interactions, forms rapidly followed by a slower transition to the fully bound state. The addition of 800 mM sodium chloride leads to a slower encounter complex formation and a decrease in electrostatic contacts. Screening of long-range electrostatic interactions could also disrupt electrostatic steering to the binding surface during the initial encounter complex formation. Simulating the binding between ApbSH3 and ArkA in the presence of salt can provide insight into the role of electrostatics in SH3 binding in general and can be compared to experimental data on AbpSH3 binding at different salt concentrations.

Identifying the STAC3/CaV1.1 interactions responsible for CaV1.1 expression in skeletal muscle.

The adaptor protein STAC3 was shown to be crucial for membrane and functional expression of the voltage sensor of EC coupling CaV1.1. In fact, CaV1.1 membrane expression is reduced and calcium currents are abolished in STAC3 KO animals. Two distinct STAC3/CaV1.1 interactions have been identified: one between the II-III loop of CaV1.1 and the SH3-1 domain of STAC3 and a second between the proximal C-terminus of CaV1.1 channels and the linker region of STAC3. Here, we investigated whether a single interaction is responsible for the CaV1.1 membrane and functional expression or if both synergistically contribute to CaV1.1 expression. To this end, we generated two STAC3 fragments, STAC3-NT, consisting of the N-terminal part, including the C1 domain and the linker region, expected to interact with the proximal C-terminus of CaV1.1, and STAC3-CT, consisting of only the SH3 domains, expected to interact with the II-III loop of CaV1.1. Expression of either one of these fragments, in a novel C2C12 STAC3 KO cell line still promoted CaV1.1 membrane expression, but at a reduced level compared to full length STAC3, suggesting that both interactions are contributing to CaV1.1 membrane expression. However, only expression of STAC3-NT supported CaV1.1 functional expression in CaV1.1 reconstituted double CaV1.1/STAC3 KO cells, indicating that only the STAC3 interaction with the proximal C-terminus is essential for CaV1.1 function in muscle cells. In addition, we analysed the ability of each STAC3 fragment to colocalize with CaV1.1 in the triads of myotubes. While the STAC3-NT fragment is still able to incorporate in CaV1.1 clusters, albeit with a 50% reduction in the Pearson's coefficient compared to full length STAC3, the STAC3-CT fragment remained diffusely localized in the cytoplasm. Experiments are planned to establish the importance of each fragment for EC coupling.

Deep-learning generative models enable design of synthetic orthologs of a signaling protein.

Understanding the relationships between protein sequence and function is critical for rational protein engineering. We have developed a deep learning model based on a variational autoencoders (VAE) to perform data-driven learning of these relationships within homologous protein families. In an application to the C-terminal SH3 domain of the high osmolarity signaling protein Sho1 (Sho1-SH3) in budding yeast, our trained VAE latent space accurately discriminates Sho1-SH3 orthologs and paralogs and enables the generative design of novel synthetic proteins predicted to possess high osmosensing function. Experimental gene synthesis and in vivo functional assays by a quantitative next-generation sequencing-based selective assay demonstrates the capability of the model to design more than 500 novel synthetic proteins possessing similar or superior function to natural sequences while sharing at minimum 60% sequence similarity with any known natural SH3 domains. Our results demonstrate the potential of deep learning models to learn the rules of protein design from extant natural proteins and use these rules to rationally design novel synthetic proteins with high functionality.

REGγ promotes mantle cell lymphoma cell apoptosis by downregulating NF-κB signaling.

Mantle cell lymphoma (MCL) is an aggressiveB-cell non-Hodgkin lymphoma (NHL). REGγ is important for tumor occurrence and development, but understanding of the specific role of REGγ in MCL is lacking. WeaimedtoidentifyREGγ effects on the proliferation and apoptosis of MCL cellsandclarifytheunderlying mechanisms. JEKO-1 cells stably transfected with a doxycycline-inducible Tet-On system expressed high levels of REGγ. JEKO-1 cells stably expressing shRNA-REGγ to reduce REGγ levels were constructed. Cell proliferation, apoptosis, and p-NF-κB, NF-κB, IkB, REGγ, p-STAT3, STAT3, and PSMB5 levels in transfected cells and in transfected cells treated with Stattic, that is a nonpeptidic small molecule exhibited to selectively inhibit signal transducer and activator of transcription factor 3 through blocking the function of its SH2 domain, were analyzed using western blotting. The proliferation of JEKO-1 cells was inhibited, and apoptosis was enhanced by increased expression of REGγ (P<0.01). REGγ inhibited MCL cell proliferation in a mouse tumor xenograft model by promoting apoptosis, increased the expression ofthe three IκB subunitsand inhibited NF-κB signaling. Overexpressed REGγ inhibited STAT3 and downregulated PSMB5 expression in MCL cells. Statticdownregulated PSMB5 and nuclear factor-kappa B (NF-κB) expressions and upregulated IκBε expression in JEKO-1 cells. We found that REGγ regulatedp-STAT3 expression by accelerating itshalf-life and downregulated the NF-κBsignaling pathway to promote MCL cell apoptosis bynegatively regulating STAT3-mediated PSMB5 expression andsubsequentlyupregulating IκB expression.

Sensitivity or specificity in protein interactions are independently regulated upon recognition: An SH2 case study.

Molecular recognition in protein-protein interactions is prompted by anchor residues that are pre-formed in a mostly bound-like ensemble before docking into their evolutionarily designed pockets. For non-competitive interactions, receptors must be sensitive to the ligand and readily bind upon recognition. On the other hand, for competitive interactions receptors must specifically distinguish among cognate ligands, limiting the free energy of the encounter complex to avoid arbitrarily locking their first approach partner. SH2 domains bind phosphorylated tyrosine (pY) sequences and use a dual pocket architecture to both specifically distinguish among ligands and auto-interact with a covalently linked ligand. Using molecular dynamics, we find that non-competitive sensors recognize sequences where pY is the anchor and can readily bind. In contrast, non-covalently linked ligands present a non-polar anchor side chain that is +2 or +3 C-terminal of pY. These distinct phenotypes define recognition by docking to separate pockets in the overall binding interface. Additionally, we show cognate interactions driven by pY anchor residues are limited to autoinhibitory sensors of multi-domain kinases, strongly suggesting the driving force for diversifying SH2 domains to bind pY residues was the arising need for an autoinhibitory sensor. Collectively, our results provide novel mechanistic insights to how the anchor ensemble can be used to dissect regulatory networks and design functional modulators targeting either sensitive or specific interactions.

In Vivo PAkt Detection and in Silico Molecular Docking to Determine the Potential of BYL as an PI3K Inhibitor to Block Growth Signal Cascade and in Breast Cancer MCF Cell Line

Comparison of genome between normal and breast cancer cell line shows that there are mutations across PI3Ka that probably cause breast cancer. PI3Ka is a kinase that plays a significant role in cell signalling transduction by phosphorylation cascade and produce an effect on cell. SH2 domain on PI3Ka binds to activated phosphor-tyrosine amino acids in growth factors, activating PI3Ka p110 catalytic domain, which recruits PIP2 to membrane and phosphorylates it to PIP3. This leads to cell signalling outcomes, including activation of PDK which phosphorylates Akt, cell proliferation and death. Protein/lipid phosphorylation levels are tightly controlled by phosphorylases and kinases; therefore, transduction levels can be turned on/off via altering kinases or phosphorylases and PI3K can be a target for treating breast cancer. However, we need find out which PI3K inhibitor (A66, TGX and unknown inhibitor BYL) shows that greatest potency and efficacy against PI3Ka with respect to pAkt in MCF-7 cell line. Here we show BYL has the potential to be a PI3Ka inhibitor and all of them inhibit pAkt. Using in silico technique of molecular docking, we explain that BYL shows the same amount of four hydrogen bonds as A66. By carrying out an experiment that generate a concentration-response curve, we demonstrate that BYL has the highest potency and efficacy with respect to pAkt level among the three inhibitors, greater than A66 which is known previously to be a potent and efficacy PI3Ka inhibitor. Our results demonstrate BYL and A66 show higher potency and efficacy than TGX; TGX with less hydrogen bonds between drug-protein interactions reveals lower potency and efficacy. Our assay accessed the inhibition of pAkt caused by three PI3Ka inhibitors (A66, TGX and BYL) in breast cancer cell line MCF-7 and found out that tumour derived stimulation of pAkt were responsive to all three inhibitors with BYL exhibits the greatest inhibition, followed by A66 and TGX in vivo due to different drug-protein interaction. Furthermore, PI3Ka inhibition has the potential to block growth signalling cascade in breast cancer and is a major target of anti-breast cancer development.

Unraveling the enthalpy of macromolecular crowding.

Concentrated solutions of synthetic polymers are often used to stabilize proteins, but there are few informed models that provide a detailed explanation of the effect. Classic crowding models are based on spherical, colloid-based solution theories, but such models only account for steric effects. To aid in the establishment of new, more informed, model, we acquired an extensive dataset that quantifies the effect of polyethylene glycol molecular weight and concentration on the temperature dependence of protein stability using 19F NMR. More specifically, we investigated the stability of the 7-kDa metastable N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) in polyethylene glycol solutions and quantified the modified standard-state free energy of unfolding and its enthalpic and entropic components. We used these data to build a new model which shows that interactions of solution components with both the folded and unfolded states must be considered. With this more nuanced understanding, we can better predict how polymers will affect the stability of protein-based drugs, how different biological molecules impact protein stability in cells and perhaps inform the design of even more useful stabilizing agents.