Publications

Activins transduce the TGF-beta pathway through a heteromeric signaling complex consisting of type I and type II receptors, and activins also inhibit bone morphogenetic protein (BMP) signaling mediated by type I receptor ALK2. Recent studies indicated that activin A cross-activates the BMP pathway through ALK2(R206H), a mutation associated with Fibrodysplasia Ossificans Progressiva (FOP). How activin A inhibits ALK2WT-mediated BMP signaling but activates ALK2(R206H)-mediated BMP signaling is not well understood, and here we offer some insights into its molecular mechanism. We first demonstrated that among four BMP type I receptors, ALK2 is the only subtype able to mediate the activin A-induced BMP signaling upon the dissociation of FKBP12. We further showed that BMP4 does not cross-signal TGF-beta pathway upon FKBP12 inhibition. In addition, although the roles of type II receptors in the ligand-independent BMP signaling activated by FOP-associated mutant ALK2 have been reported, their roles in activin A-induced BMP signaling remains unclear. We demonstrated in this study that the known type II BMP receptors contribute to activin A-induced BMP signaling through their kinase activity. Together, the current study provided important mechanistic insights at the molecular level into further understanding physiological and pathophysiological BMP signaling.

The ubiquitin ligase, Ube3a, plays important roles in brain development and functions, since its deficiency results in Angelman Syndrome (AS) while its over-expression increases the risk for autism. We previously showed that the lack of Ube3a-mediated ubiquitination of the Ca(2+)-activated small conductance potassium channel, SK2, contributes to impairment of synaptic plasticity and learning in AS mice. Synaptic SK2 levels are also regulated by protein kinase A (PKA), which phosphorylates SK2 in its C-terminal domain, facilitating its endocytosis. Here, we report that PKA activation restores theta burst stimulation (TBS)-induced long-term potentiation (LTP) in hippocampal slices from AS mice by enhancing SK2 internalization. While TBS-induced SK2 endocytosis is facilitated by PKA activation, SK2 recycling to synaptic membranes after TBS is inhibited by Ube3a. Molecular and cellular studies confirmed that phosphorylation of SK2 in the C-terminal domain increases its ubiquitination and endocytosis. Finally, PKA activation increases SK2 phosphorylation and ubiquitination in Ube3a-overexpressing mice. Our results indicate that, although both Ube3a-mediated ubiquitination and PKA-induced phosphorylation reduce synaptic SK2 levels, phosphorylation is mainly involved in TBS-induced endocytosis, while ubiquitination predominantly inhibits SK2 recycling. Understanding the complex interactions between PKA and Ube3a in the regulation of SK2 synaptic levels might provide new platforms for developing treatments for AS and various forms of autism.

Mechanosensitive Piezo1 and Piezo2 channels transduce various forms of mechanical forces into cellular signals that play vital roles in many important biological processes in vertebrate organisms. Besides mechanical forces, Piezo1 is selectively activated by micromolar concentrations of the small molecule Yoda1 through an unknown mechanism. Here, using a combination of all-atom molecular dynamics simulations, calcium imaging and electrophysiology, we identify an allosteric Yoda1 binding pocket located in the putative mechanosensory domain, approximately 40 A away from the central pore. Our simulations further indicate that the presence of the agonist correlates with increased tension-induced motions of the Yoda1-bound subunit. Our results suggest a model wherein Yoda1 acts as a molecular wedge, facilitating force-induced conformational changes, effectively lowering the channel's mechanical threshold for activation. The identification of an allosteric agonist binding site in Piezo1 channels will pave the way for the rational design of future Piezo modulators with clinical value.

A group of human mutations within the N-terminal (NT) domain of connexin 26 (Cx26) hemichannels produce aberrant channel activity, which gives rise to deafness and skin disorders, including keratitis-ichthyosis-deafness (KID) syndrome. Structural and functional studies indicate that the NT of connexin hemichannels is folded into the pore, where it plays important roles in permeability and gating. In this study, we explore the molecular basis by which N14K, an NT KID mutant, promotes gain of function. In macroscopic and single-channel recordings, we find that the N14K mutant favors the open conformation of hemichannels, shifts calcium and voltage sensitivity, and slows deactivation kinetics. Multiple copies of MD simulations of WT and N14K hemichannels, followed by the Kolmogorov-Smirnov significance test (KS test) of the distributions of interaction energies, reveal that the N14K mutation significantly disrupts pairwise interactions that occur in WT hemichannels between residue K15 of one subunit and residue E101 of the adjacent subunit (E101 being located at the transition between transmembrane segment 2 [TM2] and the cytoplasmic loop [CL]). Double mutant cycle analysis supports coupling between the NT and the TM2/CL transition in WT hemichannels, which is disrupted in N14K mutant hemichannels. KS tests of the alpha carbon correlation coefficients calculated over MD trajectories suggest that the effects of the N14K mutation are not confined to the K15-E101 pairs but extend to essentially all pairwise residue correlations between the NT and TM2/CL interface. Together, our data indicate that the N14K mutation increases hemichannel open probability by disrupting interactions between the NT and the TM2/CL region of the adjacent connexin subunit. This suggests that NT-TM2/CL interactions facilitate Cx26 hemichannel closure.

The presence of FMS-like receptor tyrosine kinase-3 internal tandem duplication (FLT3-ITD) mutations in patients with acute myeloid leukemia (AML) is associated with poor clinical outcome. FLT3 tyrosine kinase inhibitors (TKIs), although effective in kinase ablation, do not eliminate primitive FLT3-ITD(+) leukemia cells, which are potential sources of relapse. Thus, understanding the mechanisms underlying FLT3-ITD(+) AML cell persistence is essential to devise future AML therapies. Here, we show that expression of protein arginine methyltransferase 1 (PRMT1), the primary type I arginine methyltransferase, is increased significantly in AML cells relative to normal hematopoietic cells. Genome-wide analysis, coimmunoprecipitation assay, and PRMT1-knockout mouse studies indicate that PRMT1 preferentially cooperates with FLT3-ITD, contributing to AML maintenance. Genetic or pharmacological inhibition of PRMT1 markedly blocked FLT3-ITD(+) AML cell maintenance. Mechanistically, PRMT1 catalyzed FLT3-ITD protein methylation at arginine 972/973, and PRMT1 promoted leukemia cell growth in an FLT3 methylation-dependent manner. Moreover, the effects of FLT3-ITD methylation in AML cells were partially due to cross talk with FLT3-ITD phosphorylation at tyrosine 969. Importantly, FLT3 methylation persisted in FLT3-ITD(+) AML cells following kinase inhibition, indicating that methylation occurs independently of kinase activity. Finally, in patient-derived xenograft and murine AML models, combined administration of AC220 with a type I PRMT inhibitor (MS023) enhanced elimination of FLT3-ITD(+) AML cells relative to AC220 treatment alone. Our study demonstrates that PRMT1-mediated FLT3 methylation promotes AML maintenance and suggests that combining PRMT1 inhibition with FLT3 TKI treatment could be a promising approach to eliminate FLT3-ITD(+) AML cells.

To understand how protein function changes upon an allosteric perturbation, such as ligand binding and mutation, significant progress in characterizing allosteric network from molecular dynamics (MD) simulations has been made. However, determining which amino acid(s) play an essential role in the propagation of signals may prove challenging, even when the location of the source and sink is known for a protein or protein complex. This challenge is mainly due to the large fluctuations in protein dynamics that cause instability of the network topology within a single trajectory or between multiple replicas. To solve this problem, we introduce the current-flow betweenness scheme, originated from electrical network theory, to protein dynamical network analysis. To demonstrate the benefit of this new method, we chose a prototypic allosteric enzyme (IGPS or HisH-HisF dimer) as our benchmark system. Using multiple replicas of simulations and multiple network topology comparison metrics (edge ranking, path length, and node frequency), we show that the current-flow betweenness provides a significant improvement in the convergence of the allosteric networks. The improved stability of the network topology allows us to generate a delta-network between the apo and holo forms of the protein. We illustrated that the delta-network is a more rigorous way to capture the subtle changes in the networks that would otherwise be neglected by comparing node usage frequencies alone. We have also investigated the use of a linear smoothing function to improve the stability of the contact map. The methodology presented here is general and may be applied to other topology and weighting schemes. We thus conclude that, for determining protein signaling pathways between the pair(s) of source and sink, multiple MD simulation replicas are necessary, and the current-flow betweenness scheme introduced here provides a more robust approach than the geodesic scheme based on correlation edge weighting.

Relapse remains the main cause of MLL-rearranged (MLL-r) acute lymphoblastic leukemia (ALL) treatment failure resulting from persistence of drug-resistant clones after conventional chemotherapy treatment or targeted therapy. Thus, defining mechanisms underlying MLL-r ALL maintenance is critical for developing effective therapy. PRMT1, which deposits an asymmetric dimethylarginine mark on histone/non-histone proteins, is reportedly overexpressed in various cancers. Here, we demonstrate elevated PRMT1 levels in MLL-r ALL cells and show that inhibition of PRMT1 significantly suppresses leukemic cell growth and survival. Mechanistically, we reveal that PRMT1 methylates Fms-like receptor tyrosine kinase 3 (FLT3) at arginine (R) residues 972 and 973 (R972/973), and its oncogenic function in MLL-r ALL cells is FLT3 methylation dependent. Both biochemistry and computational analysis demonstrate that R972/973 methylation could facilitate recruitment of adaptor proteins to FLT3 in a phospho-tyrosine (Y) residue 969 (Y969) dependent or independent manner. Cells expressing R972/973 methylation-deficient FLT3 exhibited more robust apoptosis and growth inhibition than did Y969 phosphorylation-deficient FLT3-transduced cells. We also show that the capacity of the type I PRMT inhibitor MS023 to inhibit leukemia cell viability parallels baseline FLT3 R972/973 methylation levels. Finally, combining FLT3 tyrosine kinase inhibitor PKC412 with MS023 treatment enhanced elimination of MLL-r ALL cells relative to PKC412 treatment alone in patient-derived mouse xenografts. These results indicate that abolishing FLT3 arginine methylation through PRMT1 inhibition represents a promising strategy to target MLL-r ALL cells.

Reversible covalent inhibitors have drawn increasing attention in drug design, as they are likely more potent than noncovalent inhibitors and less toxic than covalent inhibitors. Despite those advantages, the computational prediction of reversible covalent binding presents a formidable challenge because the binding process consists of multiple steps and quantum mechanics (QM) level calculation is needed to estimate the covalent binding free energy. It has been shown that the dissociation rates and the equilibrium dissociation constants vary significantly even with similar warheads, due to noncovalent interactions. We have previously used a simplistic two-state model for predicting the relative binding selectivity of reversible covalent inhibitors ( J. Am. Chem. Soc. 2017, 139 , 17945 ). Here we go beyond binding selectivity and demonstrate that it is possible to use free energy perturbation (FEP) molecular dynamics (MD) to calculate the overall reversible covalent binding using a specially designed thermodynamic cycle. We show that FEP can predict the varying binding free energies of the analogs sharing a common warhead. More importantly, our results revealed that the chemical modification away from warhead alters the binding affinity at both noncovalent and covalent binding states, and the computational prediction can be improved by considering the binding free energy of both states. Furthermore, we explored the possibility of using a more rapid computational method, site-identification by ligand competitive saturation (SILCS), to rank the same set of reversible covalent inhibitors. We found that the fragment docking to a set of precomputed fragment maps produces a reasonable ranking. In conclusion, two independent approaches provided consistent results that the covalent binding state is suitable for the initial ranking of the reversible covalent drug candidates. For lead-optimization, the FEP approach designed here can provide more rigorous and detailed information regarding how much the covalent and noncovalent binding states are contributing to the overall binding affinity, thus offering a new avenue for fine-tuning the noncovalent interactions for optimizing reversible covalent drugs.

The connexins are members of a family of integral membrane proteins that form gap junction channels between apposed cells and/or hemichannels across the plasma membranes. The importance of the arginine at position 76 (Arg(76)) in the structure and/or function of connexin 46 (Cx46) is highlighted by its conservation across the entire connexin family and the occurrence of pathogenic mutations at this (or the corresponding homologous) residue in a number of human diseases. Two mutations at Arg(76) in Cx46 are associated with cataracts in humans, highlighting the importance of this residue. We examined the expression levels and macroscopic and single-channel properties of human Cx46 and compared them with those for two pathogenic mutants, namely R76H and R76G. To gain further insight into the role of charge at this position, we generated two additional nonnaturally occurring mutants, R76K (charge conserving) and R76E (charge inverting). We found that, when expressed exogenously in Neuro2a cells, all four mutants formed membrane hemichannels, inducing membrane permeability at levels comparable to those recorded in cells expressing the wild-type Cx46. In contrast, the number of gap-junction plaques and the magnitude of junctional coupling were reduced by all four mutations. To gain further insight into the role of Arg(76) in the function of Cx46, we performed homology modeling of Cx46 and in silico mutagenesis of Arg(76) to Gly, His, or Glu. Our studies suggest that the loss of interprotomeric interactions has a significant effect on the extracellular domain conformation and dynamics, thus affecting the hemichannel docking required for formation of cell-cell channels.

Resveratrol, a natural compound found in red wine and various vegetables, has drawn increasing interest due to its reported benefit in cardiovascular protection, neurodegenerative disorders, and cancer therapy. The mechanism by which resveratrol exerts such pleiotropic effects remains unclear. It remains as one of the most discussed polyphenol compounds in the debating "French Paradox". In this study, using molecular dynamics simulations of dipalmitoyl phosphatidylcholine (DPPC) bilayer with resveratrol, we generated a free energy map of resveratrol's location and orientation of inside the lipid bilayer. We found that resveratrol increases the surface area per lipid and decreases membrane thickness, which is the opposite effect of the well-studied cholesterol on liquid phase DPPC. Most importantly, based on the simulation observation that resveratrol has a high probability of forming hydrogen bonds with sn-1 and sn-2 ester groups, we discovered a new mechanism using experimental approach, in which resveratrol protects both sn-1 and sn-2 ester bonds of DPPC and distearoyl phosphatidylcholine (DSPC) from phospholipase A1 (PLA1) and phospholipase A2 (PLA2) cleavage. Our study elucidates the new molecular mechanism of potential health benefits of resveratrol and possibly other similar polyphenols and provides a new paradigm for drug design based on resveratrol and its analogs.