Browsing by Author "Wang, Qian"
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Item A general theoretical framework to design base editors with reduced bystander effects(Springer Nature, 2021) Wang, Qian; Yang, Jie; Zhong, Zhicheng; Vanegas, Jeffrey A.; Gao, Xue; Kolomeisky, Anatoly B.; Center for Theoretical Biological PhysicsBase editors (BEs) hold great potential for medical applications of gene therapy. However, high precision base editing requires BEs that can discriminate between the target base and multiple bystander bases within a narrow active window (4 – 10 nucleotides). Here, to assist in the design of these optimized editors, we propose a discrete-state stochastic approach to build an analytical model that explicitly evaluates the probabilities of editing the target base and bystanders. Combined with all-atom molecular dynamic simulations, our model reproduces the experimental data of A3A-BE3 and its variants for targeting the “TC” motif and bystander editing. Analyzing this approach, we propose several general principles that can guide the design of BEs with a reduced bystander effect. These principles are then applied to design a series of point mutations at T218 position of A3G-BEs to further reduce its bystander editing. We verify experimentally that the new mutations provide different levels of stringency on reducing the bystander editing at different genomic loci, which is consistent with our theoretical model. Thus, our study provides a computational-aided platform to assist in the scientifically-based design of BEs with reduced bystander effects.Item Assemblies of calcium/calmodulin-dependent kinase II with actin and their dynamic regulation by calmodulin in dendritic spines(National Academy of Sciences, 2019) Wang, Qian; Chen, Mingchen; Schafer, Nicholas P.; Bueno, Carlos; Song, Sarah S.; Hudmon, Andy; Wolynes, Peter G.; Waxham, M. Neal; Cheung, Margaret S.The structural dynamics of the dendritic synapse, arising from the remodeling of actin cytoskeletons, has been widely associated with memory and cognition. The remodeling is regulated by intracellular Ca2+ levels. Under low Ca2+ concentration, actin filaments are bundled by a calcium signaling protein, CaMKII. When the Ca2+ concentration is raised, CaMKII dissociates from actin and opens the window for actin remodeling. At present, the molecular details of the actin bundling and regulation are elusive. Herein we use experimental tools along with molecular simulations to construct a model of how CaMKII bundles actin and how the CaMKII–actin architecture is regulated by Ca2+ signals. In this way, our results explain how Ca2+ signals ultimately change the structure of the dendritic synapse.Item BAP1 is a novel regulator of HIF-1α(PNAS, 2023) Bononi, Angela; Wang, Qian; Zolondick, Alicia A.; Bai, Fang; Steele-Tanji, Mika; Suarez, Joelle S.; Pastorino, Sandra; Sipes, Abigail; Signorato, Valentina; Ferro, Angelica; Novelli, Flavia; Kim, Jin-Hee; Minaai, Michael; Takinishi, Yasutaka; Pellegrini, Laura; Napolitano, Andrea; Xu, Ronghui; Farrar, Christine; Goparaju, Chandra; Bassi, Cristian; Negrini, Massimo; Pagano, Ian; Sakamoto, Greg; Gaudino, Giovanni; Pass, Harvey I.; Onuchic, José N.; Yang, Haining; Carbone, Michele; Center for Theoretical Biological PhysicsBAP1 is a powerful tumor suppressor gene characterized by haplo insufficiency. Individuals carrying germline BAP1 mutations often develop mesothelioma, an aggressive malignancy of the serosal layers covering the lungs, pericardium, and abdominal cavity. Intriguingly, mesotheliomas developing in carriers of germline BAP1 mutations are less aggressive, and these patients have significantly improved survival. We investigated the apparent paradox of a tumor suppressor gene that, when mutated, causes less aggressive mesotheliomas. We discovered that mesothelioma biopsies with biallelic BAP1 mutations showed loss of nuclear HIF-1α staining. We demonstrated that during hypoxia, BAP1 binds, deubiquitylates, and stabilizes HIF-1α, the master regulator of the hypoxia response and tumor cell invasion. Moreover, primary cells from individuals carrying germline BAP1 mutations and primary cells in which BAP1 was silenced using siRNA had reduced HIF-1α protein levels in hypoxia. Computational modeling and co-immunoprecipitation experiments revealed that mutations of BAP1 residues I675, F678, I679, and L691 -encompassing the C-terminal domain-nuclear localization signal- to A, abolished the interaction with HIF-1α. We found that BAP1 binds to the N-terminal region of HIF-1α, where HIF-1α binds DNA and dimerizes with HIF-1β forming the heterodimeric transactivating complex HIF. Our data identify BAP1 as a key positive regulator of HIF-1α in hypoxia. We propose that the significant reduction of HIF-1α activity in mesothelioma cells carrying biallelic BAP1 mutations, accompanied by the significant reduction of HIF-1α activity in hypoxic tissues containing germline BAP1 mutations, contributes to the reduced aggressiveness and improved survival of mesotheliomas developing in carriers of germline BAP1 mutations.Item Computationally exploring the mechanism of bacteriophage T7 gp4 helicase translocating along ssDNA(National Academy of Sciences, 2022) Jin, Shikai; Bueno, Carlos; Lu, Wei; Wang, Qian; Chen, Mingchen; Chen, Xun; Wolynes, Peter G.; Gao, Yang; Center for Theoretical Biological PhysicsBacteriophage T7 gp4 helicase has served as a model system for understanding mechanisms of hexameric replicative helicase translocation. The mechanistic basis of how nucleoside 5′-triphosphate hydrolysis and translocation of gp4 helicase are coupled is not fully resolved. Here, we used a thermodynamically benchmarked coarse-grained protein force field, Associative memory, Water mediated, Structure and Energy Model (AWSEM), with the single-stranded DNA (ssDNA) force field 3SPN.2C to investigate gp4 translocation. We found that the adenosine 5′-triphosphate (ATP) at the subunit interface stabilizes the subunit–subunit interaction and inhibits subunit translocation. Hydrolysis of ATP to adenosine 5′-diphosphate enables the translocation of one subunit, and new ATP binding at the new subunit interface finalizes the subunit translocation. The LoopD2 and the N-terminal primase domain provide transient protein–protein and protein–DNA interactions that facilitate the large-scale subunit movement. The simulations of gp4 helicase both validate our coarse-grained protein–ssDNA force field and elucidate the molecular basis of replicative helicase translocation.Item Molecular mechanisms of the interhead coordination by interhead tension in cytoplasmic dyneins(National Academy of Sciences of the United States of America, 2018) Wang, Qian; Jana, Biman; Diehl, Michael R.; Cheung, Margaret S.; Kolomeisky, Anatoly B.; Onuchic, José NelsonCytoplasmic dyneins play a major role in retrograde cellular transport by moving vesicles and organelles along microtubule filaments. Dyneins are multidomain motor proteins with two heads that coordinate their motion via their interhead tension. Compared with the leading head, the trailing head has a higher detachment rate from microtubules, facilitating the movement. However, the molecular mechanism of such coordination is unknown. To elucidate this mechanism, we performed molecular dynamics simulations on a cytoplasmic dynein with a structure-based coarse-grained model that probes the effect of the interhead tension on the structure. The tension creates a torque that influences the head rotating about its stalk. The conformation of the stalk switches from the α registry to the β registry during the rotation, weakening the binding affinity to microtubules. The directions of the tension and the torque of the leading head are opposite to those of the trailing head, breaking the structural symmetry between the heads. The leading head transitions less often to the β registry than the trailing head. The former thus has a greater binding affinity to the microtubule than the latter. We measured the moment arm of the torque from a dynein structure in the simulations to develop a phenomenological model that captures the influence of the head rotating about its stalk on the differential detachment rates of the two heads. Our study provides a consistent molecular picture for interhead coordination via interhead tension.Item Molecular origin of the weak susceptibility of kinesin velocity to loads and its relation to the collective behavior of kinesins(National Academy of Sciences, 2017) Wang, Qian; Diehl, Michael R.; Jana, Biman; Cheung, Margaret S.; Kolomeisky, Anatoly B.; Onuchic, José NelsonMotor proteins are active enzymatic molecules that support important cellular processes by transforming chemical energy into mechanical work. Although the structures and chemomechanical cycles of motor proteins have been extensively investigated, the sensitivity of a motor’s velocity in response to a force is not well-understood. For kinesin, velocity is weakly influenced by a small to midrange external force (weak susceptibility) but is steeply reduced by a large force. Here, we utilize a structure-based molecular dynamic simulation to study the molecular origin of the weak susceptibility for a single kinesin. We show that the key step in controlling the velocity of a single kinesin under an external force is the ATP release from the microtubule-bound head. Only under large loading forces can the motor head release ATP at a fast rate, which significantly reduces the velocity of kinesin. It underpins the weak susceptibility that the velocity will not change at small to midrange forces. The molecular origin of this velocity reduction is that the neck linker of a kinesin only detaches from the motor head when pulled by a large force. This prompts the ATP binding site to adopt an open state, favoring ATP release and reducing the velocity. Furthermore, we show that two load-bearing kinesins are incapable of equally sharing the load unless they are very close to each other. As a consequence of the weak susceptibility, the trailing kinesin faces the challenge of catching up to the leading one, which accounts for experimentally observed weak cooperativity of kinesins motors.Item Single C-to-T substitution using engineered APOBEC3G-nCas9 base editors with minimum genome- and transcriptome-wide off-target effects(American Association for the Advancement of Science, 2020) Lee, Sangsin; Ding, Ning; Sun, Yidi; Yuan, Tanglong; Li, Jing; Yuan, Qichen; Liu, Lizhong; Yang, Jie; Wang, Qian; Kolomeisky, Anatoly B.; Hilton, Isaac B.; Zuo, Erwei; Gao, Xue; Center for Theoretical and Biological PhysicsCytosine base editors (CBEs) enable efficient cytidine-to-thymidine (C-to-T) substitutions at targeted loci without double-stranded breaks. However, current CBEs edit all Cs within their activity windows, generating undesired bystander mutations. In the most challenging circumstance, when a bystander C is adjacent to the targeted C, existing base editors fail to discriminate them and edit both Cs. To improve the precision of CBE, we identified and engineered the human APOBEC3G (A3G) deaminase; when fused to the Cas9 nickase, the resulting A3G-BEs exhibit selective editing of the second C in the 5′-CC-3′ motif in human cells. Our A3G-BEs could install a single disease-associated C-to-T substitution with high precision. The percentage of perfectly modified alleles is more than 6000-fold for disease correction and more than 600-fold for disease modeling compared with BE4max. On the basis of the two-cell embryo injection method and RNA sequencing analysis, our A3G-BEs showed minimum genome- and transcriptome-wide off-target effects, achieving high targeting fidelity.Item Theoretical Analysis of Run Length Distributions for Coupled Motor Proteins(American Chemical Society, 2019) Wang, Qian; Kolomeisky, Anatoly B.; Center for Theoretical Biological PhysicsMotor proteins, also known as biological molecular motors, play important roles in various biological processes. In recent years, properties of single-motor proteins have been intensively investigated using multiple experimental and theoretical tools. However, in real cellular systems biological motors typically function in groups, but the mechanisms of their collective dynamics remain not well understood. Here we investigate theoretically distributions of run lengths for coupled motor proteins that move along linear tracks. Our approach utilizes a method of first-passage processes, which is supplemented by Monte Carlo computer simulations. Theoretical analysis allowed us to clarify several aspects of the cooperativity mechanisms for coupled biological molecular motors. It is found that the run length distribution for two motors, in contrast to single motors, is nonmonotonic. In addition, the transport efficiency of two-motor complexes might be strongly increased. We also argue that the degree of cooperativity is influenced by several characteristics of motor proteins such as the strength of intermolecular interactions, stall forces, dissociations constants, and the detachment forces. The application of our theoretical analysis for several motor proteins is also discussed.