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1.
Adv Exp Med Biol ; 1131: 7-26, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646505

RESUMO

Measuring free Ca2+ concentration ([Ca2+]) in the cytosol or organelles is routine in many fields of research. The availability of membrane permeant forms of indicators coupled with the relative ease of transfecting cell lines with biological Ca2+ sensors have led to the situation where cellular and subcellular [Ca2+] is examined by many non-specialists. In this chapter, we evaluate the most used Ca2+ indicators and highlight what their major advantages and disadvantages are. We stress the potential pitfalls of non-ratiometric techniques for measuring Ca2+ and the clear advantages of ratiometric methods. Likely improvements and new directions for Ca2+ measurement are discussed.


Assuntos
Cálcio , Citosol , Organelas , Animais , Cálcio/metabolismo , Técnicas Citológicas , Citosol/química , Citosol/metabolismo , Humanos , Organelas/química , Organelas/metabolismo
3.
Anal Chim Acta ; 1073: 79-89, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31146839

RESUMO

We investigated the effect of oxidative stress (OS) on lipidomic perturbations in the subcellular fractions and exosomes of human embryonic kidney (HEK) 293 cells using asymmetrical flow field-flow fractionation (AF4) and nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (nUHPLC-ESI-MS/MS). We treated HEK 293 cells with hydrogen peroxide (H2O2) and fractionated the cell lysates using AF4 to determine the change in size and population of the subcellular fractions and exosomes, and to obtain narrow size fractions for lipid analysis. A total of 438 lipids from 642 identified species-including oxidized lipids-were quantified. The relative amount of secreted exosomes increased by 28% during OS, whereas the amount of subcellular species decreased by 35%. There was a significant increase in the level of oxidized phospholipids in the mitochondrion-enriched subcellular fractions, but not in the exosomes. Most high-abundance triacylglycerol (TG) species increased in the stressed cells, whereas they decreased in the exosomes. During OS, ceramides involved in the apoptotic mitochondrial pathway were accumulated in the subcellular fractions, whereas their levels were unaffected in the exosomes. The present study demonstrated that AF4 and nUHPLC-ESI-MS/MS can be used to investigate lipid alterations in subcellular and extracellular species during OS, and the pathological relationships in diseases caused by reactive oxygen species.


Assuntos
Exossomos/metabolismo , Fracionamento por Campo e Fluxo , Lipídeos/análise , Organelas/metabolismo , Estresse Oxidativo , Cromatografia Líquida de Alta Pressão , Exossomos/química , Células HEK293 , Humanos , Organelas/química , Espectrometria de Massas em Tandem
4.
Nat Chem Biol ; 15(6): 589-597, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31086330

RESUMO

To maximize a desired product, metabolic engineers typically express enzymes to high, constant levels. Yet, permanent pathway activation can have undesirable consequences including competition with essential pathways and accumulation of toxic intermediates. Faced with similar challenges, natural metabolic systems compartmentalize enzymes into organelles or post-translationally induce activity under certain conditions. Here we report that optogenetic control can be used to extend compartmentalization and dynamic control to engineered metabolisms in yeast. We describe a suite of optogenetic tools to trigger assembly and disassembly of metabolically active enzyme clusters. Using the deoxyviolacein biosynthesis pathway as a model system, we find that light-switchable clustering can enhance product formation six-fold and product specificity 18-fold by decreasing the concentration of intermediate metabolites and reducing flux through competing pathways. Inducible compartmentalization of enzymes into synthetic organelles can thus be used to control engineered metabolic pathways, limit intermediates and favor the formation of desired products.


Assuntos
Luz , Engenharia Metabólica , Redes e Vias Metabólicas/efeitos da radiação , Optogenética/métodos , Organelas/metabolismo , Organelas/efeitos da radiação , Biologia Sintética , Indóis/metabolismo , Organelas/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , Synechocystis/efeitos da radiação
5.
Anal Chim Acta ; 1068: 60-69, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31072478

RESUMO

Three hydrogen sulfide (H2S) probes based on an azonia-cyanine skeleton were successfully designed and prepared. Probe 1a, containing 4-chloro-7-nitro-1,2,3-benzoxadiazole connected to the cyanine dye, had an emission at 660 nm that was enhanced 4.5-fold by the reduced photoinduced electron transfer process when reacting with H2S. Probes 1b and 1c were constructed from cyanine dyes with electron withdrawing 2,4-dinitrophenyl and 7-nitrobenzo[c] [1,2,5]oxadiazol-4-yl groups, respectively. Probes 1b and 1c gave off-on type responses with 169- and 17-fold fluorescent enhancements at 639 nm with H2S. Their emission properties were influenced by intramolecular hydrogen bonds and intramolecular charge transfer processes. The detection limits of probes 1a-1c were calculated at 178, 121, and 9.6 nM, respectively. The intracellular imaging experiments with HeLa cells indicated probe 1a was a mitochondria-targeting H2S probe, while probes 1b and 1c were lysosome-targeting H2S probes.


Assuntos
Carbocianinas/química , Corantes Fluorescentes/química , Sulfeto de Hidrogênio/análise , Imagem Óptica , Organelas/química , Carbocianinas/síntese química , Teoria da Densidade Funcional , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Estrutura Molecular , Células Tumorais Cultivadas
6.
Proc Natl Acad Sci U S A ; 116(15): 7343-7352, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30918125

RESUMO

Mechanoreceptive organelles (MOs) are specialized subcellular entities in mechanoreceptors that transform extracellular mechanical stimuli into intracellular signals. Their ultrastructures are key to understanding the molecular nature and mechanics of mechanotransduction. Campaniform sensilla detect cuticular strain caused by muscular activities or external stimuli in Drosophila Each campaniform sensillum has an MO located at the distal tip of its dendrite. Here we analyzed the molecular architecture of the MOs in fly campaniform mechanoreceptors using electron microscopic tomography. We focused on the ultrastructural organization of NompC (a force-sensitive channel) that is linked to the array of microtubules in these MOs via membrane-microtubule connectors (MMCs). We found that NompC channels are arranged in a regular pattern, with their number increasing from the distal to the proximal end of the MO. Double-length MMCs in nompC 29+29ARs confirm the ankyrin-repeat domain of NompC (NompC-AR) as a structural component of MMCs. The unexpected finding of regularly spaced NompC-independent linkers in nompC 3 suggests that MMCs may contain non-NompC components. Localized laser ablation experiments on mechanoreceptor arrays in halteres suggest that MMCs bear tension, providing a possible mechanism for why the MMCs are longer when NompC-AR is duplicated or absent in mutants. Finally, mechanical modeling shows that upon cuticular deformation, sensillar architecture imposes a rotational activating force, with the proximal end of the MO, where more NOMPC channels are located, being subject to larger forces than the distal end. Our analysis reveals an ultrastructural pattern of NompC that is structurally and mechanically optimized for the sensory functions of campaniform mechanoreceptors.


Assuntos
Proteínas de Drosophila , Mecanorreceptores , Mecanotransdução Celular , Organelas , Canais de Receptores Transientes de Potencial , Animais , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Mecanorreceptores/química , Mecanorreceptores/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Organelas/química , Organelas/genética , Organelas/metabolismo , Canais de Receptores Transientes de Potencial/química , Canais de Receptores Transientes de Potencial/genética , Canais de Receptores Transientes de Potencial/metabolismo
7.
Science ; 363(6434)2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30923194

RESUMO

Nature regulates interference between cellular processes-allowing more complexity of life-by confining specific functions to organelles. Inspired by this concept, we designed an artificial organelle dedicated to protein engineering. We generated a membraneless organelle to translate only one type of messenger RNA-by recruiting an RNA-targeting system, stop codon-suppression machinery, and ribosomes-by means of phase separation and spatial targeting. This enables site-specific protein engineering with a tailored noncanonical function in response to one specific codon in the entire genome only in the protein of choice. Our results demonstrate a simple yet effective approach to the generation of artificial organelles that provides a route toward customized orthogonal translation and protein engineering in semisynthetic eukaryotic cells.


Assuntos
Códon/genética , Código Genético , Organelas/metabolismo , Organelas/ultraestrutura , Biossíntese de Proteínas/genética , Engenharia de Proteínas/métodos , RNA Mensageiro/genética , Animais , Células COS , Caenorhabditis elegans/genética , Membrana Celular , Cercopithecus aethiops , Células HEK293 , Humanos , Lisina/análogos & derivados , Lisina/genética , Methanosarcina , Organelas/química , RNA de Transferência/química , Ribossomos/química , Biologia Sintética
8.
Photochem Photobiol Sci ; 18(5): 1212-1217, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30834414

RESUMO

Bioluminescence is widely used in biosensors. Firefly luciferase-based bioluminescent sensors are among the most popular ones. Firefly luciferases are pH-sensitive, displaying a large red shift at acidic pH, a property that has been considered undesirable for most applications. Currently, biosensors that can detect intracellular pH are in demand, and some fluorescent biosensors are available. However, pH sensors using bioluminescence have not been used yet. Thus, we decided to harness a firefly luciferase to measure the intracellular pH in mammalian cells. For this purpose, we engineered the luciferase derived from Macrolampis sp2 firefly to localize it on the cytosol or nucleus, in order to observe pH variation in these compartments during biological activities. We first calibrated the emission ratios (R = Igreen/Ired) at different pH values. As expected, we observed a red shift of light emission under acidic conditions when the cells were subjected to different pH conditions in the presence of the K+/H+ ionophore, nigericin. Based on these results, we concluded that this firefly luciferase can be used as a diagnostic tool for measuring the intracellular pH variation in pathogenic cells or in cells during apoptosis. This is the first example of real time-monitoring of pH change using color tuning luciferase.


Assuntos
Técnicas Biossensoriais , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes , Organelas/metabolismo , Animais , Células COS , Cercopithecus aethiops , Vaga-Lumes , Concentração de Íons de Hidrogênio , Organelas/química
10.
Curr Opin Chem Biol ; 48: 81-85, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30529587

RESUMO

Identifying protein-protein interactions (PPIs) is necessary to understand the molecular mechanisms behind cellular processes. This task is complicated by the facts that many proteins can interact simultaneously (i.e. a protein complex) and may participate in more than one distinct complex. Because of this, a large number of combinatorial arrangements are possible, both of PPIs and complexes, making it a difficult task to identify all truly interacting proteins. Protein interactions also range from stable to highly transient assemblies, with lifetimes on the order of seconds [1]. Therefore, studies identifying PPIs must not only contend with the arrangement of proteins into PPIs and complexes, but the stability of the interactions as well. Because of the difficulty of the task, many approaches have been used to identify and study the dynamics of PPIs. In this review, we will summarize a number of the techniques currently used to identify protein-protein interactions, with a focus on recent developments.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Animais , Biotinilação , Humanos , Organelas/química , Organelas/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas , Proteínas/análise , Proteômica/métodos
11.
Cell Mol Life Sci ; 75(21): 3907-3929, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30066087

RESUMO

Effects of macromolecular crowding on structural and functional properties of ordered proteins, their folding, interactability, and aggregation are well documented. Much less is known about how macromolecular crowding might affect structural and functional behaviour of intrinsically disordered proteins (IDPs) or intrinsically disordered protein regions (IDPRs). To fill this gap, this review represents a systematic analysis of the available literature data on the behaviour of IDPs/IDPRs in crowded environment. Although it was hypothesized that, due to the excluded-volume effects present in crowded environments, IDPs/IDPRs would invariantly fold in the presence of high concentrations of crowding agents or in the crowded cellular environment, accumulated data indicate that, based on their response to the presence of crowders, IDPs/IDPRs can be grouped into three major categories, foldable, non-foldable, and unfoldable. This is because natural cellular environment is not simply characterized by the presence of high concentration of "inert" macromolecules, but represents an active milieu, components of which are engaged in direct physical interactions and soft interactions with target proteins. Some of these interactions with cellular components can cause (local) unfolding of query proteins. In other words, since crowding can cause both folding and unfolding of an IDP or its regions, the outputs of the placing of a query protein to the crowded environment would depend on the balance between these two processes. As a result, and because of the spatio-temporal heterogeneity in structural organization of IDPs, macromolecular crowding can differently affect structures of different IDPs. Recent studies indicate that some IDPs are able to undergo liquid-liquid-phase transitions leading to the formation of various proteinaceous membrane-less organelles (PMLOs). Although interiors of such PMLOs are self-crowded, being characterized by locally increased concentrations of phase-separating IDPs, these IDPs are minimally foldable or even non-foldable at all (at least within the physiologically safe time-frame of normal PMLO existence).


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Substâncias Macromoleculares/química , Conformação Proteica , Dobramento de Proteína , Proteínas Intrinsicamente Desordenadas/genética , Modelos Moleculares , Organelas/química , Organelas/genética
12.
World J Microbiol Biotechnol ; 34(9): 139, 2018 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-30151754

RESUMO

The mechanisms of heavy metal resistance in microbial cells involve multiple pathways. They include the formation of complexes with specific proteins and other compounds, the excretion from the cells via plasma membrane transporters in case of procaryotes, and the compartmentalization of toxic ions in vacuoles, cell wall and other organelles in case of eukaryotes. The relationship between heavy metal tolerance and inorganic polyphosphate metabolism was demonstrated both in prokaryotic and eukaryotic microorganisms. Polyphosphates, being polyanions, are involved in detoxification of heavy metals through complex formation and compartmentalization. The bacteria and fungi cultivated in the presence of some heavy metal cations contain the enhanced levels of polyphosphate. In bacteria, polyphosphate sequesters heavy metals; some of metal cations stimulate an exopolyphosphatase activity, which releases phosphate from polyphosphates, and MeHPO4- ions are then transported out of the cells. In fungi, the overcoming of heavy metal stresses is associated with the accumulation of polyphosphates in cytoplasmic inclusions, vacuoles and cell wall and the formation of cation/polyphosphate complexes. The effects of knockout mutations and overexpression of the genes encoding polyphosphate-metabolizing enzymes on heavy metal resistance are discussed.


Assuntos
Tolerância a Medicamentos/fisiologia , Inativação Metabólica/fisiologia , Metais Pesados/metabolismo , Polifosfatos/metabolismo , Hidrolases Anidrido Ácido/metabolismo , Archaea/fisiologia , Bactérias/metabolismo , Transporte Biológico , Cátions/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Tolerância a Medicamentos/genética , Células Eucarióticas/patologia , Fungos/fisiologia , Metais Pesados/toxicidade , Organelas/química , Fosfatos/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Células Procarióticas/fisiologia
13.
J Biosci ; 43(3): 437-445, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30002263

RESUMO

Long-distance transport of many organelles inside eukaryotic cells is driven by the dynein and kinesin motors on microtubule filaments. More than 30 years since the discovery of these motors, unanswered questions include motor- organelle selectivity, structural determinants of processivity, collective behaviour of motors and track selection within the complex cytoskeletal architecture, to name a few. Fluorescence microscopy has been invaluable in addressing some of these questions. Here we present a review of some efforts to understand these sub-microscopic machines using fluorescence.


Assuntos
Dineínas/metabolismo , Células Eucarióticas/metabolismo , Cinesina/metabolismo , Microscopia de Fluorescência/métodos , Microtúbulos/metabolismo , Miosinas/metabolismo , Animais , Anticorpos/química , Bioensaio , Transporte Biológico , Dineínas/química , Células Eucarióticas/química , Células Eucarióticas/ultraestrutura , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinesina/química , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Microscopia de Fluorescência/instrumentação , Microtúbulos/química , Microtúbulos/ultraestrutura , Miosinas/química , Imagem Óptica/instrumentação , Imagem Óptica/métodos , Organelas/química , Organelas/metabolismo , Organelas/ultraestrutura
14.
Talanta ; 186: 406-412, 2018 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-29784380

RESUMO

This work reports an approach for imaging the localization, distribution, and level of miRNA in different organelles based on an activated fluorescence signal triggered by an alteration of the specific binding-induced conformation of the designed activatable probe. We selected miR-150 as an miRNA example to image its localization, distribution, and level in human cervical cancer cells (HeLa cells). The results indicate that miR-150 is localized and distributed in different subcellular organelles (mainly in mitochondria and lysosomes) and that its levels (actually its concentrations) in lysosomes are higher than those in mitochondria in both HeLa and MCF-7 cells. Moreover, the level of miRNA in cells is displayed in a height-dependent (in z-direction) manner. This approach can also be used to image the localization and distribution of various miRNAs (such as miR-150 and miR-214) in different organelles in cancer cells simultaneously. The probes exhibit high resistance to cellular endo- and exonucleases, with high specificity; the capability of avoiding false signals, with a high signal-to-background ratio; and a good ability to operate in complicated environments. The developed approach may provide a useful tool for studying the localization and distribution and evaluating the level of multiple tumor-related miRNAs in cells.


Assuntos
MicroRNAs/análise , Imagem Óptica , Organelas/química , Linhagem Celular Tumoral , Fluorescência , Células HeLa , Humanos , Células MCF-7 , Espectrometria de Fluorescência
15.
Nature ; 557(7707): 679-683, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29769721

RESUMO

Non-membrane-bound organelles such as nucleoli, processing bodies, Cajal bodies and germ granules form by the spontaneous self-assembly of specific proteins and RNAs. How these biomolecular condensates form and interact is poorly understood. Here we identify two proteins, ZNFX-1 and WAGO-4, that localize to Caenorhabditis elegans germ granules (P granules) in early germline blastomeres. Later in germline development, ZNFX-1 and WAGO-4 separate from P granules to define an independent liquid-like condensate that we term the Z granule. In adult germ cells, Z granules assemble into ordered tri-condensate assemblages with P granules and Mutator foci, which we term PZM granules. Finally, we show that one biological function of ZNFX-1 and WAGO-4 is to interact with silencing RNAs in the C. elegans germline to direct transgenerational epigenetic inheritance. We speculate that the temporal and spatial ordering of liquid droplet organelles may help cells to organize and coordinate the complex RNA processing pathways that underlie gene-regulatory systems, such as RNA-directed transgenerational epigenetic inheritance.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Epigênese Genética/genética , Organelas/química , Organelas/metabolismo , Interferência de RNA , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/enzimologia , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Células Germinativas/metabolismo , RNA Helicases/metabolismo
16.
J Trace Elem Med Biol ; 48: 172-180, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29773177

RESUMO

The functions performed by the ER are diverse: synthesis of steroid hormones, synthesis of proteins for the plasma membrane, lysosomes, as well as proteins meant for exocytosis, protein folding, formation of disulfide bonds, N-linked glycosylation, etc. Selenoproteins localized in this organelle are definitely involved in the processes occurring in it, and the most common of them include participation in protein degradation, regulation of ER stress and redox metabolism. ER stress has been registered in many types of cancer cells. The ability to persist under prolonged ER stress increases their survival, resistance to drugs and immunity. Disturbances in the redox regulation of the cell cycle, which result in the accumulation of misfolded proteins in the ER, viral infection, disruption of Ca2+ regulation, are known to cause an evolutionarily conserved reaction - unfolded protein response (UPR) and, ultimately, lead to ER stress. Since selenoproteins, as oxidoreductases, possess antioxidant properties, and their role in the regulation of important processes, such as carcinogenesis and ER stress, has been actively studied in the recent decades, the subject of this review is highly relevant.


Assuntos
Carcinogênese/química , Retículo Endoplasmático/química , Organelas/química , Selenoproteínas/análise , Animais , Cálcio/análise , Cálcio/metabolismo , Carcinogênese/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Organelas/metabolismo , Selenoproteínas/metabolismo , Estresse Fisiológico
17.
Biochemistry ; 57(17): 2405-2414, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29683665

RESUMO

A flurry of research in recent years has revealed the molecular origins of many membraneless organelles to be the liquid phase separation of intrinsically disordered proteins (IDPs). Consequently, protein disorder has emerged as an important driver of intracellular compartmentalization by providing specialized microenvironments chemically distinct from the surrounding medium. Though the importance of protein disorder and its relationship to intracellular phase behavior are clear, a detailed understanding of how such phase behavior can be predicted and controlled remains elusive. While research in IDPs has largely focused on the implications of structural disorder on cellular function and disease, another field, that of artificial protein polymers, has focused on the de novo design of protein polymers with controllable material properties. A subset of these polymers, specifically those derived from structural proteins such as elastin and resilin, are also disordered sequences that undergo liquid-liquid phase separation. This phase separation has been used in a variety of biomedical applications, and researchers studying these polymers have developed methods to precisely characterize and tune their phase behavior. Despite their disparate origins, both fields are complementary as they study the phase behavior of intrinsically disordered polypeptides. This Perspective hopes to stimulate collaborative efforts by highlighting the similarities between these two fields and by providing examples of how such collaboration could be mutually beneficial.


Assuntos
Compartimento Celular/genética , Microambiente Celular/genética , Proteínas Intrinsicamente Desordenadas/genética , Organelas/genética , Membrana Celular/química , Membrana Celular/genética , Elastina/química , Elastina/genética , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas Intrinsicamente Desordenadas/química , Organelas/química , Peptídeos/química , Peptídeos/genética , Polímeros , Conformação Proteica
18.
Biochemistry ; 57(18): 2590-2596, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29671583

RESUMO

We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light-inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.


Assuntos
Dimerização , Endopeptidases/química , Tetra-Hidrofolato Desidrogenase/química , Compartimento Celular/efeitos dos fármacos , Compartimento Celular/genética , Compartimento Celular/efeitos da radiação , Luz , Organelas/química , Organelas/efeitos da radiação , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Transporte Proteico/efeitos da radiação , Sirolimo/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Tetra-Hidrofolato Desidrogenase/efeitos da radiação
19.
Biochemistry ; 57(17): 2470-2477, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29569441

RESUMO

Compartmentalization of biochemical components, interactions, and reactions is critical for the function of cells. While intracellular partitioning of molecules via membranes has been extensively studied, there has been an expanding focus in recent years on the critical cellular roles and biophysical mechanisms of action of membraneless organelles (MLOs) such as the nucleolus. In this context, a substantial body of recent work has demonstrated that liquid-liquid phase separation plays a key role in MLO formation. However, less is known about MLO dissociation, with phosphorylation being the primary mechanism demonstrated thus far. In this Perspective, we focus on another mechanism for MLO dissociation that has been described in recent work, namely a reentrant phase transition (RPT). This concept, which emerges from the polymer physics field, provides a mechanistic basis for both formation and dissolution of MLOs by monotonic tuning of RNA concentration, which is an outcome of cellular processes such as transcription. Furthermore, the RPT model also predicts the formation of dynamic substructures (vacuoles) of the kind that have been observed in cellular MLOs. We end with a discussion of future directions in terms of open questions and methods that can be used to answer them, including further exploration of RPTs in vitro, in cells, and in vivo using ensemble and single-molecule methods as well as theory and computation. We anticipate that continued studies will further illuminate the important roles of reentrant phase transitions and associated non-equilibrium dynamics in the spatial patterning of the biochemistry and biology of the cell.


Assuntos
Fenômenos Biofísicos , Compartimento Celular/genética , Organelas/genética , Transição de Fase , Citoplasma/química , Citoplasma/genética , Membranas/química , Simulação de Dinâmica Molecular , Organelas/química , Fosforilação
20.
Biochemistry ; 57(17): 2499-2508, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29509422

RESUMO

Liquid-liquid phase separation and related condensation processes of intrinsically disordered proteins (IDPs), proteins with intrinsically disordered regions, and nucleic acids underpin various condensed-liquid droplets or gel-like assemblies in the cellular environment. Collectively referred to as condensates, these bodies provide spatial/temporal compartmentalization, often serving as hubs for regulated biomolecular interactions. Examples include certain extracellular materials, transcription complexes, and membraneless organelles such as germ and stress granules and the nucleolus. They are critically important to cellular function; thus misregulation of their assembly is implicated in many diseases. Biomolecular condensates are complex entities. Our understanding of their inner workings is only in its infancy. Nonetheless, insights into basic biophysical principles of their assembly can be gained by applying analytical theories to elucidate how IDP phase behaviors are governed by the properties of the multivalent, solvent-mediated interactions entailed by the proteins' amino acid sequences. Here we briefly review the background of the pertinent polymer theories and outline the approximations that enable a tractable theoretical account of the dependence of IDP phase behaviors on the charge pattern of the IDP sequence. Of relevance to the homeostatic assembly of compositionally and functionally distinct condensates in the cellular context, theory indicates that the propensity for populations of different IDP sequences to mix or demix upon phase separation is affected by the similarity or dissimilarity of the sequence charge patterns. We also explore prospects of extending analytical theories to account for dynamic aspects of biomolecular condensates and to incorporate effects of cation-π, π-π, and temperature-dependent hydrophobic interactions on IDP phase properties.


Assuntos
Sequência de Aminoácidos/genética , Proteínas Intrinsicamente Desordenadas/genética , Ácidos Nucleicos/genética , Organelas/genética , Fenômenos Bioquímicos , Interações Hidrofóbicas e Hidrofílicas , Proteínas Intrinsicamente Desordenadas/química , Modelos Moleculares , Ácidos Nucleicos/química , Organelas/química , Transição de Fase , Temperatura Ambiente
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