Light-Induced Dimerization Approaches to Control Cellular Processes

Light-inducible approaches provide a means to control biological systems with spatial and temporal resolution that is unmatched by traditional genetic perturbations. Recent developments of optogenetic and chemo-optogenetic systems for induced proximity in cells facilitate rapid and reversible manipulation of highly dynamic cellular processes and have become valuable tools in diverse biological applications. New expansions of the toolbox facilitate control of signal transduction, genome editing, “painting” patterns of active molecules onto cellular membranes, and light-induced cell cycle control. A combination of light- and chemically induced dimerization approaches have also seen interesting progress. Herein, an overview of optogenetic systems and emerging chemo-optogenetic systems is provided, and recent applications in tackling complex biological problems are discussed.     

Development and applications of the ABEEM fluctuating charge molecular force field in the ion-containing systems

The microscopic mechanisms of ion hydration and ion selectivity in biomolecular systems are long-standing research topics,in which the difficulty is how to reasonably and accurately describe the ion-water and ion-biomolecule interactions.This paper summarizes the development and applications of the atom-bond electronegativity equalization fluctuating charge force field model,ABEEM/MM,in the investigations of ion hydration,metalloproteins and ion-DNA bases systems.Based on high-level quantum chemistry calculations,the parameters were optimized and the molecular potential functions were constructed and applied to studies of structures,activities,energetics,and thermodynamic and kinetic properties of these ion-containing systems.The results show that the performance of ABEEM/MM is generally better than that of the common force fields,and its accuracy can reach or approach that of the high-level ab initio MP2 method.These studies provide a solid basis for further investigations of ion selectivity in biomolecular systems,the structures and properties of metalloproteins and other related ion-containing systems.     

Photocleavable proteins that undergo fast and efficient dissociation

Photocleavable molecules can enable the light-dependent modulation of biomolecular activities with high spatiotemporal precision. We have previously reported a photocleavable protein (PhoCl1) that, uniquely, is a fully genetically encoded photocleavable molecule that can be introduced into cells in the form of its corresponding gene to enable optogenetic control of biomolecular activities. However, the first generation PhoCl1 exhibited a relatively slow rate of dissociation, potentially limiting its utility. Here, we report the X-ray crystal structures of the PhoCl1 green state, red state, and cleaved empty barrel. Molecular dynamics (MD) simulations were performed to provide insight into the precise dissociation mechanism. Using structure-guided engineering and directed evolution, we have developed PhoCl2c with higher contrast ratio and PhoCl2f with faster dissociation. We characterized the performance of these new variants as purified proteins and in cultured cells. Our results demonstrate that PhoCl2 variants exhibit faster and more efficient dissociation, which should enable improved optogenetic manipulations of protein localization and protein–protein interactions in living cells.

Photocleavable proteins can enable the light-dependent modulation of biomolecular activities with high spatiotemporal precision.     

Identification of Casiopeina II-gly secondary targets through a systems pharmacology approach

Casiopeinas are a group of copper-based compounds designed to be used as less toxic, more efficient chemotherapeutic agents. In this study, we analyzed the in vitro effects of Casiopeina II-gly on the expression of canonical biological pathways. Using microarray data from HeLa cell lines treated with Casiopeina II-gly, we identified biological pathways that are perturbed after treatment. We present a novel approach integrating pathway analysis and network theory: The Pathway Crosstalk Network. We constructed a network with deregulated pathways, featuring links between those pathways that crosstalk with each other. We identified modules grouping deregulated pathways that are functionally related. Through this approach, we were able to identify three features of Casiopeina treatment: (a) Perturbation of signaling pathways, related to induction of apoptosis; (b) perturbation of metabolic pathways, and (c) activation of immune responses. These findings can be useful to drive new experimental exploration on their role in adverse effects and efficacy of Casiopeinas.     

Modern optical microscopy methods to study biomolecular condensates

Cells achieve highly intricate internal organization via membrane-bound and membraneless organelles. Research over the past decade has implicated liquid–liquid phase separation, a phenomenon by which charged and often disordered biological macromolecules assemble into reversible liquid-like condensates, as the mechanism of formation of membraneless organelles in cells. During the same period, optical microscopy saw exciting advancements, including the super-resolution revolution, that were quickly adopted by researchers in the biological community. Today, there exists a vast library of techniques capable of providing unprecedented information regarding the formation, function, and dynamics of biomolecular condensates. In this review, we discuss a select number of modern optical microscopy methods that are particularly suited for studying biomolecular condensates both in vitro and in cells, as well as the associated technical challenges.     

Mechanistic studies of nitrite reactions with metalloproteins and models relevant to mammalian physiology

This review provides a summary of reaction mechanisms involving the interactions of nitrite ion with metal centers relevant to physiology. The majority of the systems that have been investigated are heme proteins and models, where nitrite reacts with the central metal ions to generate important iron-NO_x intermediates and subsequent NO_x products. We also discuss reactions with other potentially relevant systems. Nitrite is formed as a product of NO autoxidation in aqueous media and can be formed by the bacterial reduction of ingested nitrate as well. It is now generally accepted that under certain conditions nitrite, which is present in mammalian fluids and tissue at micromolar concentrations, can serve as a biological reserve of the bioregulatory agent nitric oxide. However, it is possible that nitrite serves other functions as well. The goal of this review is to evaluate the present state of understanding regarding these pathways and the delicate interplay between nitrite and the various NO_x species of biological relevance.     

Direct Observation of Kinetic Pathways of Biomolecular Recognition

The pathways of molecular recognition, which is a central event in all biological processes, belong to the most important subjects of contemporary research in biomolecular science. By using fluorescence spectroscopy in a microfluidics channel, it can be determined that molecular recognition of α‐chymotrypsin in hydrous surroundings at two different pH values (3.6 and 6.3) follows two distinctly different pathways. Whereas one corroborates an induced‐fit model (pH 3.6), the other one (pH 6.3) is consistent with the selected‐fit model of biomolecular recognition. The role of massive structural perturbations of differential recognition pathways could be ruled out by earlier XRD studies, rather was consistent with the femtosecond‐resolved observation of dynamic flexibility of the protein at different pH values. At low concentrations of ligands, the selected‐fit model dominates, whereas increasing the ligand concentration leads to the induced‐fit model. From molecular modelling and experimental results, the timescale associated with the conformational flexibility of the protein plays a key role in the selection of a pathway in biomolecular recognition.     

Dynamic spatial and structural organization in artificial cells regulates signal processing by protein scaffolding

Structural and spatial organization are fundamental properties of biological systems that allow cells to regulate a wide range of biochemical processes. This organization is often transient and governed by external cues that initiate dynamic self-assembly processes. The construction of synthetic cell-like materials with similar properties requires the hierarchical and reversible organization of selected functional components on molecular scaffolds to dynamically regulate signaling pathways. The realization of such transient molecular programs in synthetic cells, however, remains underexplored due to the associated complexity of such hierarchical platforms. In this contribution, we effectuate dynamic spatial organization of effector protein subunits in a synthetic biomimetic compartment, a giant unilamellar vesicle (GUV), by associating in a reversible manner two fragments of a split luciferase to the membrane. This induces their structural dimerization, which consequently leads to the activation of enzymatic signaling. Importantly, such organization and activation are dynamic processes, and can be autonomously regulated – thus opening up avenues toward continuous spatiotemporal control over supramolecular organization and signaling in an artificial cell.

Engineered artificial cells respond to environmental cues through a pre-programmed enzymatic machinery that induces spatio-structural organization and activation of effector proteins at the lipid membrane.     

Oxidative Stress and Multi-Organel Damage Induced by Two Novel Phytocannabinoids,CBDB and CBDP,in Breast Cancer Cells

Over the last few years, much attention has been paid to phytocannabinoids derived from Cannabis for their therapeutic potential. Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and cannabidiol (CBD) are the most abundant compounds of the Cannabis sativa L. plant. Recently, novel phytocannabinoids, such as cannabidibutol (CBDB) and cannabidiphorol (CBDP), have been discovered. These new molecules exhibit the same terpenophenolic core of CBD and differ only for the length of the alkyl side chain. Roles of CBD homologs in physiological and pathological processes are emerging but the exact molecular mechanisms remain to be fully elucidated. Here, we investigated the biological effects of the newly discovered CBDB or CBDP, compared to the well-known natural and synthetic CBD (nat CBD and syn CBD) in human breast carcinoma cells that express CB receptors. In detail, our data demonstrated that the treatment of cells with the novel phytocannabinoids affects cell viability, increases the production of reactive oxygen species (ROS) and activates cellular pathways related to ROS signaling, as already demonstrated for natural CBD. Moreover, we observed that the biological activity is significantly increased upon combining CBD homologs with drugs that inhibit the activity of enzymes involved in the metabolism of endocannabinoids, such as the monoacylglycerol lipase (MAGL) inhibitor, or with drugs that induces the activation of cellular stress pathways, such as the phorbol ester 12-myristate 13-acetate (PMA).     

Multiple pathways in conformational transitions of the alanine dipeptide: an application of dynamic importance sampling

We present multiple dynamic transition pathways on the two-dimensional dihedral plane between conformational states of the alanine dipeptide. The method used in this study is dynamic importance sampling (DIMS). To perform DIMS, unbiased molecular dynamic simulations are used to generate equilibrium ensembles for the alanine dipeptide within different states. Free energy surfaces on the dihedral plane are calculated from the equilibrium simulations, and four energy minima defined from the surface are used as the starting and ending points for DIMS dynamics. The DIMS method represents an important step towards finding multiple transition pathways within complex biomolecular systems.     

Small-molecule fluorescent probes for imaging gaseous signaling molecules: current progress and future implications

Endogenous gaseous signaling molecules including nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H₂S) have been demonstrated to perform significant physiological and pharmacological functions and are associated with various diseases in biological systems. In order to obtain a deeper insight into their roles and mechanisms of action, it is desirable to develop novel techniques for effectively detecting gaseous signaling molecules. Small-molecule fluorescent probes have been proven to be a powerful approach for the detection and imaging of biological messengers by virtue of their non-invasiveness, high selectivity, and real-time in situ detection capability. Based on the intrinsic properties of gaseous signaling molecules, numerous fluorescent probes have been constructed to satisfy various demands. In this perspective, we summarize the recent advances in the field of fluorescent probes for the detection of NO, CO and H₂S and illustrate the design strategies and application examples of these probes. Moreover, we also emphasize the challenges and development directions of gasotransmitter-responsive fluorescent probes, hoping to provide a general implication for future research.

This perspective article aims to introduce the design principles and recognition strategies of small-molecule fluorescent probes which are applied for the detection of gas signaling molecules including NO, CO and H₂S in biological systems.     

Exercise training modulates the gut microbiota profile and impairs inflammatory signaling pathways in obese children

Childhood obesity has reached epidemic levels and is a serious health concern associated with metabolic syndrome, nonalcoholic fatty liver disease, and gut microbiota alterations. Physical exercise is known to counteract obesity progression and modulate the gut microbiota composition. This study aims to determine the effect of a 12-week strength and endurance combined training program on gut microbiota and inflammation in obese pediatric patients. Thirty-nine obese children were assigned randomly to the control or training group. Anthropometric and biochemical parameters, muscular strength, and inflammatory signaling pathways in mononuclear cells were evaluated. Bacterial composition and functionality were determined by massive sequencing and metabolomic analysis. Exercise reduced plasma glucose levels and increased dynamic strength in the upper and lower extremities compared with the obese control group. Metagenomic analysis revealed a bacterial composition associated with obesity, showing changes at the phylum, class, and genus levels. Exercise counteracted this profile, significantly reducing the Proteobacteria phylum and Gammaproteobacteria class. Moreover, physical activity tended to increase some genera, such as Blautia, Dialister, and Roseburia, leading to a microbiota profile similar to that of healthy children. Metabolomic analysis revealed changes in short-chain fatty acids, branched-chain amino acids, and several sugars in response to exercise, in correlation with a specific microbiota profile. Finally, the training protocol significantly inhibited the activation of the obesity-associated NLRP3 signaling pathway. Our data suggest the existence of an obesity-related deleterious microbiota profile that is positively modified by physical activity intervention. Exercise training could be considered an efficient nonpharmacological therapy, reducing inflammatory signaling pathways induced by obesity in children via microbiota modulation.Subject terms: High-throughput screening, DNA metabolism     

Photocatalysis in Chemical Biology: Extending the Scope of Optochemical Control and Towards New Frontiers in Semisynthetic Bioconjugates and Biocatalysis

Biomedical research has been empowered by tools that enable spatial and temporal control of biological systems. These have predominantly come from photocaged bioactive molecules (optochemical control; N. Ankenbruck, T. Courtney, Y. Naro, A. Deiters, Angew. Chem. Int. Ed. 2018 , 57, 2768–2798) and light-dependent proteins (optogenetic control; L. Fenno, O. Yizhar, K. Deisseroth, Annu. Rev. Neurosci. 2011 , 34, 389–412). Recent advances in photocatalysis offer the opportunity to amplify these strategies by providing new dimension of biorthogonality. Photocatalysis also empowers bioconjugation with unprecedented reactivities enabling new crosslinking chemistry or biomolecule functionalization, while merging photocatalysis with biocatalysis extends the scope of both of these powerful classes of transformation.     

Domain Analysis and Motif Matcher (DAMM): A Program to Predict Selectivity Determinants in Monosiga brevicollis PDZ Domains Using Human PDZ Data

Choanoflagellates are single-celled eukaryotes with complex signaling pathways. They are considered the closest non-metazoan ancestors to mammals and other metazoans and form multicellular-like states called rosettes. The choanoflagellate Monosiga brevicollis contains over 150 PDZ domains, an important peptide-binding domain in all three domains of life (Archaea, Bacteria, and Eukarya). Therefore, an understanding of PDZ domain signaling pathways in choanoflagellates may provide insight into the origins of multicellularity. PDZ domains recognize the C-terminus of target proteins and regulate signaling and trafficking pathways, as well as cellular adhesion. Here, we developed a computational software suite, Domain Analysis and Motif Matcher (DAMM), that analyzes peptide-binding cleft sequence identity as compared with human PDZ domains and that can be used in combination with literature searches of known human PDZ-interacting sequences to predict target specificity in choanoflagellate PDZ domains. We used this program, protein biochemistry, fluorescence polarization, and structural analyses to characterize the specificity of A9UPE9_MONBE, a M. brevicollis PDZ domain-containing protein with no homology to any metazoan protein, finding that its PDZ domain is most similar to those of the DLG family. We then identified two endogenous sequences that bind A9UPE9 PDZ with <100 μM affinity, a value commonly considered the threshold for cellular PDZ–peptide interactions. Taken together, this approach can be used to predict cellular targets of previously uncharacterized PDZ domains in choanoflagellates and other organisms. Our data contribute to investigations into choanoflagellate signaling and how it informs metazoan evolution.     

Flicker noise spectroscopy and its application: Information hidden in chaotic signals (review)

Fundamentals of flicker noise spectroscopy (FNS), a general phenomenological approach to analyzing dynamics and structure of complex nonlinear systems by extracting information contained in chaotic signals of diverse nature generated by such systems, are presented. The primary idea of FNS is to disclose information hidden in correlation links which are present in a sequence of various irregularities (spikes, jumps, discontinuities in derivatives of different orders) that occur in the measured dynamic variables at all levels of spatiotemporal hierarchy of systems under study. The information is derived from power spectra S(f) (f, frequency) and transient difference moments Φ^(p)(τ)(τ, time delay parameter) of different orders p. The procedures of averaging over time interval T, which are introduced in FNS when computing S(f) and Φ^(p)(τ), differ from the procedures of averaging in the Gibbs approach. In the latter case, due to the adoption of the ergodic hypothesis, the average values of dynamic quantities over time are replaced by the average values of the same quantities over a statistical ensemble. It came to pass that the Φ^(p)(τ) functions are formed exclusively by jumps of a dynamic variable on different spatiotemporal levels of the system’s hierarchy, whereas the formation of S(f) is contributed to by spikes and jumps. The informative parameters extracted from S(f) and Φ^(p)(τ) describe correlation times and characterize loss of “memory” (correlation links) in these correlation time intervals for the “spike” and “jump” irregularities. These parameters can be determined using the expressions derived for the case of steady-state evolution. Here the “steady state” implies an evolution state that is characterized by the same values of informative parameters on every level of the system’s hierarchy. In contrast to the scaling self-similarity in theory of fractals and renormgroup, FNS introduces a multiparametric self-similarity for the S(f) and Φ^(p)(τ) functions, which is generally characterized by a set of parameters rather than one scaling factor. The S(f) and Φ⁽²⁾(τ) functions which are related to different types of information may be viewed, in the steady-state case, as fluctuation-dissipation relations that complement each other informatively. Examples of such generalized relations are presented for fluctuations of electric voltage under open-circuit conditions (the Nyquist theorem), the Levy diffusion, the hydrodynamic fluctuations at fully developed turbulence, and the flicker noise fluctuations of the electric current density in electron-conducting systems. To analyze the dynamics of non-steady processes, formulas are presented for calculating nonstationarity indicators (factors) and estimating time instants when most noticeable changes occur in systems under study, including those preceding catastrophic evolution changes. The studies carried out to date show that the FNS method can be used in solving problems of three types. The first is the determination of parameters or patterns that characterize the dynamics or specific features of structural organization of open complex systems. The second involves search for precursors of sharpest changes in the states of various open dissipative systems on the basis of available information conceming the dynamics of such systems. And the third problem concerns the dynamics of redistribution of perturbations in distributed systems. It is solved by analyzing dynamic correlations in chaotic signals that are measured simultaneously at different points in space. The review demonstrates some applications of the FNS methodology. In particular, it considers some physicochemical and natural processes the data for which were obtained from electrochemical and biological measurements.     

近红外上转换纳米转换器在光遗传学调控中的应用

光遗传学作为一种新兴的生物技术,能够在时间和空间上精准调控生理功能。尤其是在基于视紫红质离子通道蛋白来操控神经兴奋性及钙信号通路激活等方面,近年来该技术吸引了广泛的关注。然而,目前该技术所使用的光遗传学工具只能被可见光激发,难以穿透深层组织并实现无创地光学调控。为了解决这个问题,最近一些研究通过使用稀土掺杂上转换纳米粒子作为光转换器,将组织可穿透的近红外光转化为可见光发射,从而使复杂活体条件下的光遗传学调控成为可能。我们对近年来上转换纳米粒子介导的光遗传学技术的开发和应用进展做了详细的总结。另外,关于未来如何进一步推进该技术可用于临床研究提出了建议和展望。     

Photochemical control of bacterial gene expression based on trans encoded genetic switches

Controlling gene expression by light with fine spatiotemporal resolution not only allows understanding and manipulating fundamental biological processes but also fuels the development of novel therapeutic strategies. In complement to exploiting optogenetic tools, photochemical strategies mostly rely on the incorporation of photo-responsive small molecules into the corresponding biomacromolecular scaffolds. Therefore, generally large synthetic effort is required and the switching of gene expression in both directions within a single system remains a challenge. Here, we report a trans encoded ribo-switch, which consists of an engineered tRNA mimicking structure (TMS), under control of small photo-switchable signalling molecules. The signalling molecules consist of two amino glycoside molecules that are connected via an azobenzene unit. The light responsiveness of our system originates from the photo-switchable noncovalent interactions between the signalling molecule and the TMS switch, leading to the demonstration of photochemically controlled expression of two different genes. We believe that this modular design will provide a powerful platform for controlling the expression of other functional proteins with high spatiotemporal resolution employing light as a stimulus.

Controlling gene expression by light with fine spatiotemporal resolution not only allows understanding and manipulating fundamental biological processes but also fuels the development of novel therapeutic strategies.     

Computational analysis revealed miRNAs produced by Chikungunya virus target genes associated with antiviral immune responses and cell cycle regulation

Chikungunya virus (CHIKV) that causes chikungunya fever, is an alphavirus that belongs to the Togaviridae family containing a single-stranded RNA genome. Mosquitoes of the Aedes species act as the vectors for this virus and can be found in the blood, which can be passed from an infected person to a mosquito through mosquito bites. CHIKV has drawn much attention recently because of its potential of causing an epidemic. As the detailed mechanism of its pathogenesis inside the host system is still lacking, in this in silico research we have hypothesized that CHIKV might create miRNAs, which would target the genes associated with host cellular regulatory pathways, thereby providing the virus with prolonged refuge. Using bioinformatics approaches we found several putative miRNAs produced by CHIKV. Then we predicted the genes of the host targeted by these miRNAs. Functional enrichment analysis of these targeted genes shows the involvement of several biological pathways regulating antiviral immune stimulation, cellular proliferation, and cell cycle, thereby provide themselves with prolonged refuge and facilitate their pathogenesis, which in turn may lead to disease conditions. Finally, we analyzed a publicly available microarray dataset (GSE49985) to determine the altered expression levels of the targeted genes and found genes associated with pathways such as cell differentiation, phagocytosis, T-cell activation, response to cytokine, autophagy, Toll-like receptor signaling, RIG-I like receptor signaling and apoptosis. Our finding presents novel miRNAs and their targeted genes, which upon experimental validation could facilitate in developing new therapeutics to combat CHIKV infection and minimize CHIKV mediated diseases.     

Design and synthesis of novel pinacolylboronate containing combretastatin ‘antimitotic agent’ analogues

We developed a procedure to synthesize pinacolyl boronate containing stilbene derivatives and used this procedure to synthesize boron-containing combretastatin analogues. The key step involves the Wittig reaction of the ylide 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboratophenyl)-methyl triphenylphosphonium bromide 11 with 3,4,5-trimethoxy benzaldehyde in the presence of ^tBuONa in DMF, providing 88% yield. We are now in a position to evaluate the biological activity of these derivatives as modulators of TGF-beta signaling pathways.