Biotin-painted proteins have thermodynamic stability switched by kinetic folding routes.

Biotin-labeled proteins are widely used as tools to study protein-protein interactions and proximity in living cells. Proteomic methods broadly employ proximity-labeling technologies based on protein biotinylation in order to investigate the transient encounters of biomolecules in subcellular compartments. Biotinylation is a post-translation modification in which the biotin molecule is attached to lysine or tyrosine residues. So far, biotin-based technologies proved to be effective instruments as affinity and proximity tags. However, the influence of biotinylation on aspects such as folding, binding, mobility, thermodynamic stability, and kinetics needs to be investigated. Here, we selected two proteins [biotin carboxyl carrier protein (BCCP) and FKBP3] to test the influence of biotinylation on thermodynamic and kinetic properties. Apo (without biotin) and holo (biotinylated) protein structures were used separately to generate all-atom structure-based model simulations in a wide range of temperatures. Holo BCCP contains one biotinylation site, and FKBP3 was modeled with up to 23 biotinylated lysines. The two proteins had their estimated thermodynamic stability changed by altering their energy landscape. In all cases, after comparison between the apo and holo simulations, differences were observed on the free-energy profiles and folding routes. Energetic barriers were altered with the density of states clearly showing changes in the transition state. This study suggests that analysis of large-scale datasets of biotinylation-based proximity experiments might consider possible alterations in thermostability and folding mechanisms imposed by the attached biotins.

Pericytes enable effective angiogenesis in the presence of proinflammatory signals.

Angiogenesis frequently occurs in the context of acute or persistent inflammation. The complex interplay of proinflammatory and proangiogenic cues is only partially understood. Using an experimental model, permitting exposure of developing blood vessel sprouts to multiple combinations of diverse biochemical stimuli and juxtacrine cell interactions, we present evidence that a proinflammatory cytokine, tumor necrosis factor (TNF), can have both proangiogenic and antiangiogenic effects, depending on the dose and the presence of pericytes. In particular, we find that pericytes can rescue and enhance angiogenesis in the presence of otherwise-inhibitory high TNF doses. This sharp switch from proangiogenic to antiangiogenic effect of TNF observed with an escalating dose of this cytokine, as well as the effect of pericytes, are explained by a mathematical model trained on the biochemical data. Furthermore, this model was predictive of the effects of diverse combinations of proinflammatory and antiinflammatory cues, and variable pericyte coverage. The mechanism supports the effect of TNF and pericytes as modulating signaling networks impinging on Notch signaling and specification of the Tip and Stalk phenotypes. This integrative analysis elucidates the plasticity of the angiogenic morphogenesis in the presence of diverse and potentially conflicting cues, with immediate implications for many physiological and pathological settings.

Toward understanding cancer stem cell heterogeneity in the tumor microenvironment.

The epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) formation are two paramount processes driving tumor progression, therapy resistance, and cancer metastasis. Recent experiments show that cells with varying EMT and CSC phenotypes are spatially segregated in the primary tumor. The underlying mechanisms generating such spatiotemporal dynamics in the tumor microenvironment, however, remain largely unexplored. Here, we show through a mechanism-based dynamical model that the diffusion of EMT-inducing signals such as TGF-ß, together with noncell autonomous control of EMT and CSC decision making via the Notch signaling pathway, can explain experimentally observed disparate localization of subsets of CSCs with varying EMT phenotypes in the tumor. Our simulations show that the more mesenchymal CSCs lie at the invasive edge, while the hybrid epithelial/mesenchymal (E/M) CSCs reside in the tumor interior. Further, motivated by the role of Notch-Jagged signaling in mediating EMT and stemness, we investigated the microenvironmental factors that promote Notch-Jagged signaling. We show that many inflammatory cytokines such as IL-6 that can promote Notch-Jagged signaling can (i) stabilize a hybrid E/M phenotype, (ii) increase the likelihood of spatial proximity of hybrid E/M cells, and (iii) expand the fraction of CSCs. To validate the predicted connection between Notch-Jagged signaling and stemness, we knocked down JAG1 in hybrid E/M SUM149 human breast cancer cells in vitro. JAG1 knockdown significantly restricted tumor organoid formation, confirming the key role that Notch-Jagged signaling can play in tumor progression. Together, our integrated computational-experimental framework reveals the underlying principles of spatiotemporal dynamics of EMT and CSCs.

Uncovering the molecular mechanisms behind disease-associated leptin variants.

The pleiotropic hormone leptin has a pivotal role in regulating energy balance by inhibiting hunger and increasing energy expenditure. Homozygous mutations found in the leptin gene are associated with extreme obesity, marked hyperphagia, and impaired immune function. Although these mutations have been characterized in vivo, a detailed understanding of how they affect leptin structure and function remains elusive. In the current work, we used NMR, differential scanning calorimetry, molecular dynamics simulations, and bioinformatics calculations to characterize the effects of these mutations on leptin structure and function and binding to its cognate receptor. We found that mutations identified in patients with congenital leptin deficiency not only cause leptin misfolding or aggregation, but also cause changes in the dynamics of leptin residues on the receptor-binding interface. Therefore, we infer that mutation-induced leptin deficiency may arise from several distinct mechanisms including (i) blockade of leptin receptor interface II, (ii) decreased affinity in the second step of leptin's interaction with its receptor, (iii) leptin destabilization, and (iv) unsuccessful threading through the covalent loop, leading to leptin misfolding/aggregation. We propose that this expanded framework for understanding the mechanisms underlying leptin deficiency arising from genetic mutations may be useful in designing therapeutics for leptin-associated disorders.

Understanding the Principles of Pattern Formation Driven by Notch Signaling by Integrating Experiments and Theoretical Models.

Notch signaling is an evolutionary conserved cell-cell communication pathway. Besides regulating cell-fate decisions at an individual cell level, Notch signaling coordinates the emergent spatiotemporal patterning in a tissue through ligand-receptor interactions among transmembrane molecules of neighboring cells, as seen in embryonic development, angiogenesis, or wound healing. Due to its ubiquitous nature, Notch signaling is also implicated in several aspects of cancer progression, including tumor angiogenesis, stemness of cancer cells and cellular invasion. Here, we review experimental and computational models that help understand the operating principles of cell patterning driven by Notch signaling. First, we discuss the basic mechanisms of spatial patterning via canonical lateral inhibition and lateral induction mechanisms, including examples from angiogenesis, inner ear development and cancer metastasis. Next, we analyze additional layers of complexity in the Notch pathway, including the effect of varying cell sizes and shapes, ligand-receptor binding within the same cell, variable binding affinity of different ligand/receptor subtypes, and filopodia. Finally, we discuss some recent evidence of mechanosensitivity in the Notch pathway in driving collective epithelial cell migration and cardiovascular morphogenesis.

The Pierced Lasso Topology Leptin has a Bolt on Dynamic Domain Composed by the Disordered Loops I and III.

Leptin is an important signaling hormone, mostly known for its role in energy expenditure and satiety. Furthermore, leptin plays a major role in other proteinopathies, such as cancer, marked hyperphagia, impaired immune function, and inflammation. In spite of its biological relevance in human health, there are no NMR resonance assignments of the human protein available, obscuring high-resolution characterization of the soluble protein and/or its conformational dynamics, suggested as being important for receptor interaction and biological activity. Here, we report the nearly complete backbone resonance assignments of human leptin. Chemical shift-based secondary structure prediction confirms that in solution leptin forms a four-helix bundle including a pierced lasso topology. The conformational dynamics, determined on several timescales, show that leptin is monomeric, has a rigid four-helix scaffold, and a dynamic domain, including a transiently formed helix. The dynamic domain is anchored to the helical scaffold by a secondary hydrophobic core, pinning down the long loops of leptin to the protein body, inducing motional restriction without a well-defined secondary or tertiary hydrogen bond stabilized structure. This dynamic region is well suited for and may be involved in functional allosteric dynamics upon receptor binding.

A Biophysical Model Uncovers the Size Distribution of Migrating Cell Clusters across Cancer Types.

Migration from the primary tumor is a crucial step in the metastatic cascade. Cells with various degrees of adhesion and motility migrate and are launched into the bloodstream as single circulating tumor cells (CTC) or multicellular CTC clusters. The frequency and size distributions of these clusters have been recently measured, but the underlying mechanisms enabling these different modes of migration remain poorly understood. We present a biophysical model that couples the phenotypic plasticity enabled by the epithelial-mesenchymal transition (EMT) and cell migration to explain the modes of individual and collective cancer cell migration. This reduced physical model captures how cells undergo a transition from individual migration to collective cell migration and robustly recapitulates CTC cluster fractions and size distributions observed experimentally across several cancer types, thus suggesting the existence of common features in the mechanisms underlying cancer cell migration. Furthermore, we identify mechanisms that can maximize the fraction of CTC clusters in circulation. First, mechanisms that prevent a complete EMT and instead increase the population of hybrid epithelial/mesenchymal (E/M) cells are required to recapitulate CTC size distributions with large clusters of 5 to 10 cells. Second, multiple intermediate E/M states give rise to larger and heterogeneous clusters formed by cells with different epithelial-mesenchymal traits. Overall, this biophysical model provides a platform to continue to bridge the gap between the molecular and biophysical regulation of cancer cell migration and highlights that a complete EMT might not be required for metastasis. SIGNIFICANCE: A biophysical model of cancer cell invasion integrates phenotypic heterogeneity and cell migration to interpret experimental observations of circulating tumor cell clusters and provides new predictions.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/21/5527/F1.large.jpg.

Quantitative Characteristic of ncRNA Regulation in Gene Regulatory Networks.

RNA is mostly known for its role in protein synthesis, where it encodes information for protein sequence in its messenger RNA (mRNA) form (translation). Yet, RNA molecules regulate several cellular processes other than translation. Here, we present an overview of several mathematical models that help understanding and characterizing the role of noncoding RNA molecules (ncRNAs) in regulating gene expression and protein synthesis. First, we discuss relatively simple models where ncRNAs can modulate protein synthesis via targeting a mRNA. Then, we consider the case of feedback interactions between ncRNAs and their target proteins, and discuss several biological applications where these feedback architectures modulate a cellular phenotype and control the levels of intrinsic and extrinsic noise. Building from these simple circuit motifs, we examine feed-forward circuit motifs involving ncRNAs that generate precise spatial and temporal patterns of protein expression. Further, we investigate the competition between ncRNAs and other endogenous RNA molecules and show that the cross talk between coding and noncoding RNAs can form large genetic circuits that involve up to hundreds of chemical species. Finally, we discuss the role of ncRNAs in modulating cell-cell signaling pathways and therefore the dynamics of spatiotemporal pattern formation in a tissue.

A mechanism-based computational model to capture the interconnections among epithelial-mesenchymal transition, cancer stem cells and Notch-Jagged signaling.

Epithelial-mesenchymal transition (EMT) and cancer stem cell (CSCs) formation are two fundamental and well-studied processes contributing to cancer metastasis and tumor relapse. Cells can undergo a partial EMT to attain a hybrid epithelial/mesenchymal (E/M) phenotype or a complete EMT to attain a mesenchymal one. Similarly, cells can reversibly gain or lose 'stemness'. This plasticity in cell states is modulated by signaling pathways such as Notch. However, the interconnections among the cell states enabled by EMT, CSCs and Notch signaling remain elusive. Here, we devise a computational model to investigate the coupling among the core decision-making circuits for EMT, CSCs and Notch. Our model predicts that hybrid E/M cells are most likely to associate with stem-like traits and enhanced Notch-Jagged signaling - a pathway implicated in therapeutic resistance. Further, we show that the position of the 'stemness window' on the 'EMT axis' is varied by altering the coupling strength between EMT and CSC circuits, and/or modulating Notch signaling. Finally, we analyze the gene expression profile of CSCs from several cancer types and observe a heterogeneous distribution along the 'EMT axis', suggesting that different subsets of CSCs may exist with varying phenotypes along the epithelial-mesenchymal axis. We further investigate therapeutic perturbations such as treatment with metformin, a drug associated with decreased cancer incidence and increased lifespan of patients. Our mechanism-based model explains how metformin can both inhibit EMT and blunt the aggressive potential of CSCs simultaneously, by driving the cells out of a hybrid E/M stem-like state with enhanced Notch-Jagged signaling.

Theory of protein folding.

Protein folding should be complex. Proteins organize themselves into specific three-dimensional structures, through a myriad of conformational changes. The classical view of protein folding describes this process as a nearly sequential series of discrete intermediates. In contrast, the energy landscape theory of folding considers folding as the progressive organization of an ensemble of partially folded structures through which the protein passes on its way to the natively folded structure. As a result of evolution, proteins have a rugged funnel-like landscape biased toward the native structure. Connecting theory and simulations of minimalist models with experiments has completely revolutionized our understanding of the underlying mechanisms that control protein folding.

Pierced Lasso Topology Controls Function in Leptin.

Protein engineering is a powerful tool in drug design and therapeutics, where disulphide bridges are commonly introduced to stabilize proteins. However, these bonds also introduce covalent loops, which are often neglected. These loops may entrap the protein backbone on opposite sides, leading to a "knotted" topology, forming a so-called Pierced Lasso (PL). In this elegant system, the "knot" is held together with a single disulphide bridge where part of the polypeptide chain is threaded through. The size and position of these covalent loops can be manipulated through protein design in vitro, whereas nature uses polymorphism to switch the PL topology. The PL protein leptin shows genetic modification of an N-terminal residue, adding a third cysteine to the same sequence. In an effort to understand the mechanism of threading of these diverse topologies, we designed three loop variants to mimic the polymorphic sequence. This adds elegance to the system under study, as it allows the generation of three possible covalent loops; they are the original wild-type C-terminal loop protein, the fully circularized unthreaded protein, and the N-terminal loop protein, responsible for different lasso topologies. The size of the loop changes the threading mechanism from a slipknotting to a plugging mechanism, with increasing loop size. Interestingly, the ground state of the native protein structure is largely unaffected, but biological assays show that the activity is maximized by properly controlled dynamics in the threaded state. A threaded topology with proper conformational dynamics is important for receptor interaction and activation of the signaling pathways in vivo.

Energy landscape along an enzymatic reaction trajectory: hinges or cracks?

Are the most dynamically flexible regions around the equilibrium structure of an enzyme the same regions involved in the transition state for rate limiting processes involved in the enzymatic reaction? Kern and-coworkers (Wolf-Watzet al., 2004; Henzler-Wildman et al., 2007a, 2007b) have shown that insights about functionally relevant motions that determine the overall enzyme turnover rate can be obtained by investigating conformational dynamics around the equilibrium basin of the enzyme adenylate kinase. An allosteric change in protein structure turns out to be the controlling process. In this commentary we compare results of this study with earlier predictions of the route by which the enzyme undergoes its conformational change. These predictions are based on the idea that the energy surface for the protein is determined by the end structures of the conformational change. A key issue is whether the protein moves by specific hinges or whether it "cracks" and accesses partially unfolded states during its structural change.

Dynamics of electron transfer pathways in cytochrome C oxidase.

Cytochrome c oxidase mediates the final step of electron transfer reactions in the respiratory chain, catalyzing the transfer between cytochrome c and the molecular oxygen and concomitantly pumping protons across the inner mitochondrial membrane. We investigate the electron transfer reactions in cytochrome c oxidase, particularly the control of the effective electronic coupling by the nuclear thermal motion. The effective coupling is calculated using the Green's function technique with an extended Huckel level electronic Hamiltonian, combined with all-atom molecular dynamics of the protein in a native (membrane and solvent) environment. The effective coupling between Cu(A) and heme a is found to be dominated by the pathway that starts from His(B204). The coupling between heme a and heme a(3) is dominated by a through-space jump between the two heme rings rather than by covalent pathways. In the both steps, the effective electronic coupling is robust to the thermal nuclear vibrations, thereby providing fast and efficient electron transfer.

Specific and nonspecific collapse in protein folding funnels.

Experiments with fast folding proteins are beginning to address the relationship between collapse and folding. We investigate how different scenarios for folding can arise depending on whether the folding and collapse transitions are concurrent or whether a nonspecific collapse precedes folding. Many earlier studies have focused on the limit in which collapse is fast compared to the folding time; in this work we focus on the opposite limit where, at the folding temperature, collapse and folding occur simultaneously. Real proteins exist in both of these limits. The folding mechanism varies substantially in these two regimes. In the regime of concurrent folding and collapse, nonspecific collapse now occurs at a temperature below the folding temperature (but slightly above the glass transition temperature).