Andrew Harris, Rutgers University
Hosted by Sergei Sukharev
Title: Molecular Navigation Through Connexin Channels: First Steps
Time: 4:00PM - 5:00PM
Date: Monday, February 4, 2019
Abstract
Connexin proteins form wide pores in plasma membrane and between adjacent cells. These gated pores are permeable to atomic ions and small molecules, thereby mediating electrical and molecular signaling. The intercellular channels (“gap junction channels”) permit cytosolic molecules such as IP3 and cAMP to pass between cells. The plasma membrane channels (“hemichannels”) play autocrine/paracrine roles by releasing molecules such as glutamate and ATP into the extracellular environment. The molecular signaling mediated by connexin channels is of critical biomedical importance, being intimately involved in development, normal physiology, and response to trauma and disease. Defects in connexin channels cause human pathologies. Although connexin channels as a class are permeable to a wide variety of small molecules, channels formed by each of the 21 human connexin isoforms have strikingly different ionic and molecular selectivities. The molecular permselectivity is not a simple function of pore width but depends on as yet uncharacterized interactions between specific permeants and the pore lumen of each connexin isoform; there is little correlation among channel unitary conductance, limiting pore diameter and/or charge selectivity. To help elucidate the mechanisms that influence and define selective molecular permeation, molecular dynamics simulations were applied to similar two molecules – one permeant and one not – in a connexin hemichannel. The results highlight issues and factors that come into play in selective molecular permeation of wide pores that are different from those that dominate permeation of atomic ions through ion-selective pores. These include mechanisms of selectivity involving low-energy interactions, and ‘‘permeant’’ and side-chain flexibility, orientation and anisotropy. A key element of the energetic landscape is the entropic contribution due to molecules that can occupy many configurations and orientations in a non-rigid water-filled pore. Pore width influences the energetic landscapes experienced by these molecules and differences between them, but other factors are strongly involved. Also, for both the permeant and impermeant test molecules, the computed energetic barriers extend through most of the pore, without significant binding (energy wells). The results suggest that this type of analysis may be useful in exploring the molecular basis by which connexin channels distinguish among (potential) permeating molecules, and how mutations may alter the permeation process.
Ruth Nussinov, NCI/NIH
Hosted by David Fushman
Title: Allosteric regulatory mechanisms in the Ras signaling network
Time: 4:00PM - 5:00PM
Date: Monday, February 11, 2019
Abstract
How do Ras isoforms attain oncogenic specificity at the membrane? Oncogenic KRas, HRas, and NRas differentially populate distinct cancers. How they selectively activate effectors and why is KRas4B the most prevalent are highly significant questions. We also ask how Ras activates its effectors, including Raf and PI3K lipid kinase, which are also among the most highly mutated proteins in cancer. Despite decades of studies, major mechanistic questions are still unanswered. Broadly, we aim to figure out the hallmarks of oncogenic signaling in the cell.
Gevorg Grigoryan, Dartmouth College
Hosted by Garegin Papoian
Title: The “Digital” Nature of Protein Structure
Time: 4:00PM - 5:00PM
Date: Monday, February 18, 2019
Abstract
While we know that protein sequence encodes structure, capturing this sequence-to-structure mapping computationally has been difficult. Particularly so because the space of structural possibilities appears immense and complex. We propose that this space should nevertheless be describable as a combination of discrete local structural patterns. We introduce the concept of a TERM (tertiary motif), which encapsulates the full structural environment around a given residue, and show that the protein structural universe is highly degenerate at the level of TERMs. In fact, only 650 TERMs describe over 50% of the structural database at sub-Angstrom resolution. We go on to show that such degeneracy enables the direct quantification of sequence-structure relationships. Local sequence models can be extracted for each TERM contained in a protein structure, based on the frequent reuse of TERMs in unrelated proteins, with the overall protein structure described as a combination of these models. We have begun to demonstrate the broad applicability of such a framework across a variety of applications: 1) protein design: we have either partially or fully redesigned multiple proteins using TERM data alone, as well as designed novel structures de novo (with experimental validation); 2) structure prediction: we found that TERM-based sequence statistics identify accurate models; 3) we have shown that mutational stability changes are predicted quantitatively from TERM data alone. Earlier findings of degeneracies in the protein structure (e.g., for secondary and super-secondary motifs), have greatly advanced computational structural biology. TERM-based mining of structural data is the next logical step that should provide further quantitative insights into sequence-structural relationships.
Jinwoo Lee, UMD
Hosted by Sergei Sukharev
Title: Ebola virus glycoprotein spike recruits cholesterol for efficient fusion
Time: 4:00PM - 5:00PM
Date: Monday, February 25, 2019
Abstract
Cholesterol serves critical roles in membrane fusion of envelope viruses by modulating physical properties such as thickness and curvature of biological membrane. Ebola virus (EBOV) belongs to the class 1 virus with HIV and Influenza and shares similar structural features. The membrane fusion of EBOV requires internal receptor Niemann-Pick C1, which is cholesterol transporter, reside in late endosomal compartment and budding occurs in the plasma membrane where a relatively high concentration of cholesterol is located. Thus, the importance of cholesterol in EBOV membrane fusion needs to be characterized. Here we present that EBOV glycoprotein membrane proximal external region/transmembrane (MPER/TM) domain recruits cholesterol to local environments for efficient fusion. The fusion and virus-like particles (VLPs) entry assays show that cholesterol concentration in the viral membrane is more critical for efficient fusion event to occur. Cholesterol titration experiments using NMR revealed that G660 is critical for cholesterol interaction. The G660L mutant shows the wider angle between MPER and TM compare to WT in DEER experiments and lost interaction with cholesterol in NMR experiments. The total internal reflection fluorescence (TIRF) microscopy experiment shows that cholesterol in viral membrane enhances fusion kinetics and the probability of full fusion events. The G660L mutant shows higher hemifusion probability and also exhibited a cell entry efficiency of only about 25% of WT VLPs. Furthermore, the cholesterol-lowering drug, statin was tested in VLPs entry assay and statin treated VLPs entry efficiency was decreased compare to untreated. Taken together, glycoprotein and cholesterol interaction is important for efficient membrane fusion of EBOV and cholesterol in the viral membrane is more critical in EBOV membrane fusion and statin as potential EBOV treatment cocktail ingredient.
John Biddle, Harvard University
Hosted by Mikhail Anisimov/Sergei Sukharev
Title: Negative Reciprocity, Not Ordered Assembly, Underlies the Interaction of Sox2 and Oct4 on DNA
Time: 4:00PM - 5:00PM
Date: Monday, March 11, 2019
Abstract
Single-molecule data on the transcription factors Sox2 and Oct4 publication five years ago as evidence of ordered assembly on DNA. The quantity cited in support of this conclusion, however, is not a measure of ordered assembly. Moreover, these single-molecule experiments aim to infer what takes place at DNA binding loci by following transcription factors, and this inference requires a more in-depth biophysical analysis than had been carried out. We performed such an analysis and did not find support for the hypothesis of ordered assembly, but we did find something novel: that the expression of Sox2 in these cells increased genomic binding by Oct4, while the expression of Oct4 decreased genomic binding by Sox2. We call this surprising phenomenon “negative reciprocity”, and show that it cannot be accounted for at thermodynamic equilibrium with only one kind of Sox2-Oct4 binding loci. Either the cell must expend energy so as to maintain the system away from thermodynamic equilibrium, or Sox2 and Oct4 must bind at diverse genomic loci in such a way that at some of these loci they assist each other in binding, while at others they hinder each other. The analytical techniques used to derive these results are of general interest, and increasingly so as single-molecule tracking techniques continue to develop.
Quan Qing, Arizona State University
Hosted by Wolfgang Losert
Title: Precisely regulate ERK signaling pathway with local electric fields
Time: 4:00PM - 5:00PM
Date: Monday, April 8, 2019
Abstract
Our main research interest is to understand how artificial electronics can interact with biological systems. Live cells rely on a big network of signaling pathways that sense and respond to biochemical, electrical and mechanical (BEM) stimuli. We want to explore if we can modulate this BEM network, particularly with external electric field, and investigate the mechanism at the molecular level. In addition, we develop new strategies enabled by nanomaterials/nanostructures for best integration of artificial electronic devices with live cells so that we can achieve higher sensitivity with better biocompatibility for in vitro and in vivo applications. In this presentation I will talk about our recent discovery of modulation of extracellular-signal-regulated kinase (ERK) pathway using alternative current (AC) electric fields (EFs). The amplitude, duration, and frequency of activation of the ERK pathway code diverse spectrum of information at cell, tissue and organism levels to instruct cells to migrate, proliferate, or differentiate. Synchronized control of ERK activation would provide a powerful approach to regulate cell behaviors. Here we show for the first time that AC EFs in a new frequency range can reproducibly activate ERK activities through patterned local microelectrodes with single-cell resolution. Both the amplitude and frequency of ERK activation can be precisely synchronized and modulated. We pinpointed a new mechanism of AC EF induced highly specific phosphorylation of epidermal growth factor (EGF) receptor (EGFR) to activate the EGFR-ERK pathway, which may serve as a powerful platform for control of cell behaviors with implications in wide range of biomedical applications.
Haribabu Arthanari, Harvard University
Hosted by Kwaku Dayie
Title: Functional roles of low complexity regions in the human proteome
Time: 4:00PM - 5:00PM
Date: Monday, April 15, 2019
Abstract
Structural biology studies over the last 50 years have provided an unprecedented view of structured domains, yet the structural details of a significant portion of the expressed proteome, characterized by IDRs, still remain in the dark. Research over the past decade has brought the functional significance of IDRs into the limelight. There are several established cases where disordered regions of a protein and IDPs transition to a structured state when they encounter a binding partner. Some of these interactions are weak, yet critical, dictated by need for a transient signal. Key cellular processes, including transcription and translation are orchestrated by many such interactions. The business end of transcription factors, the transactivation domain (TAD), is usually disordered and assume structure upon binding “helper” proteins such as co-activators and mediators to regulate transcription. The presentation will detail a structure-based approach to unravel the molecular details the interaction between the TAD and a Mediator subunit and use the information to target the interaction in two clinically relevant systems, affecting multiple drug resistance and lipogenesis.
Edward O'Brien, Pennsylvania State University
Hosted by Garegin Papoian
Title: Non-equilibrium coupling of protein structure and function to translation elongation kinetics
Time: 4:00PM - 5:00PM
Date: Monday, April 22, 2019
Abstract
Protein folding research has been dominated by the assumption that thermodynamics determines protein structure and function. And that when the folding process is compromised in vivo the proteostasis machinery — chaperones, deaggregases, the proteasome — work to restore proteins to their soluble, functional form or degrade them to maintain the cellular pool of proteins in a quasi-equilibrium state. During the past decade, however, more and more proteins have been identified for which altering only their speed of synthesis alters their structure and function, the efficiency of the down-stream processes they take part in, and cellular phenotype. Indeed, evidence has emerged that evolutionary selection pressures have encoded translation-rate information into mRNA molecules to coordinate diverse co-translational processes. Thus, non-equilibrium physics can play a fundamental role in influencing nascent protein behavior, mRNA sequence evolution, and disease. In this talk I will discuss my lab's efforts to understand and model this phenomenon through the development application of theoretical tools from the physical sciences including coarse-grained simulations, chemical kinetics and statistical mechanics.
Michael Feig, Michigan State University
Hosted by Pratyush Tiwary
Title: Crowding, Clustering, and Phase Transitions in Crowded Cellular Environments
Time: 4:00PM - 5:00PM
Date: Monday, April 29, 2019
Abstract
Biological macromolecules function in dense, crowded cellular environments. Early studies of crowding effects have emphasized volume exclusion effects, but it is becoming clear that frequent non-specific interactions between proteins, nucleic acids, and metabolites may be the more important factor in modulating the structure and dynamics of biomolecules. Computer simulation studies at different scales of a series of models ranging from concentrated homogeneous protein solutions to models of bacterial cytoplasms are presented to explore the effects of non-specific quinary protein-protein interactions on protein stability and dynamics. One focus is on the formation of transient clusters that determine diffusive properties and lead to liquid-liquid phase transitions. The computational results are related to existing experimental data and the challenges and opportunities to expand the current studies to whole-cell modeling in molecular detail are discussed.
Yun Chen, Johns Hopkins University
Hosted by Arpita Upadhyaya
Title: Force-dependent extracellular matrix remodeling in the tumor microenvironment alters exosome diffusion and induces CAF phenotypes
Time: 4:00PM - 5:00PM
Date: Monday, May 6, 2019
Abstract
Carcinoma-associated fibroblasts (CAFs) can be detected during early stages of cancer development in tumor stroma. CAFs are often associated with high-grade malignancies of poor prognosis in breast cancer and other squamous cell carcinomas by secreting cytokines promoting cancer cells to replicate, metastasize and resist apoptosis. Understanding the mechanisms by which normal stroma-dwelling cells transform to CAFs might inspire strategies to suppress CAF induction and improve prognosis. Normal fibroblasts (NFs) in stroma can be induced to CAFs by exosomes containing microRNA and cytokines, which are secreted by cancer cells. However, it is not clear how these CAF-inducing factors secreted by cancer cells can reach stromal NFs on the other side of the basement membrane (BM) in vivo: At the early stages of breast cancer, cancer cells do not exhibit high expression of metalloproteases (MMPs) to digest extracellular matrix (ECM), which imposes a diffusion barrier for CAF-inducing factors to reach stroma. Microscopically, the diffusion barrier is formed by the laminin/fibronectin fibrils in the BM and collagen fibrils in the stroma. Moreover, at early stages cancer cells are not yet metastatic and cannot migrate into the proximity of stromal NFs. Despite the lack of physical proximity and low MMP secretion, we observed a series of force-dependent events initiated by cancer cells which might enable CAF induction. First, cancer cells use the strong forces to mechanically remodel the ECM fibril organization in the tumor microenvironment to enhance the diffusion of CAF-inducing factors. Second, cancer cells exert the forces to directly activate mechanosignaling pathways in the stromal fibroblasts to induce CAF phenotypes. In particular, we observed that the diffusion rate of CAF-inducing factors correlates with the degree of ECM fibril alignment, and is important in CAF induction. We found that it is early-stage breast cancer cells which align the ECM fibrils so that the diffusion rate is increased. Furthermore, we observed that ECM remodeling was associated with long-range (>1000 μm) mechanical forces collectively generated by epithelial spheroids. Overall, we conclude that cancer cells of epithelial origin can facilitate CAF induction, through long-range mechanical forces, by decreasing diffusion barrier across BM.