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1.
Biochem Biophys Res Commun ; 715: 150008, 2024 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-38685186

RESUMO

In the last decade, much attention was given to the study of physiological amyloid fibrils. These structures include A-bodies, which are the nucleolar fibrillar formations that appear in the response to acidosis and heat shock, and disassemble after the end of stress. One of the proteins involved in the biogenesis of A-bodies, regardless of the type of stress, is Von-Hippel Lindau protein (VHL). Known also as a tumor suppressor, VHL is capable to form amyloid fibrils both in vitro and in vivo in response to the environment acidification. As with most amyloidogenic proteins fusion with various tags is used to increase the solubility of VHL. Here, we first performed AFM-study of fibrils formed by VHL protein and by VHL fused with GST-tag (GST-VHL) at acidic conditions. It was shown that formed by full-length VHL fibrils are short heterogenic structures with persistent length of 2400 nm and average contour length of 409 nm. GST-tag catalyzes VHL amyloid fibril formation, superimpose chirality, increases length and level of hierarchy, but decreases rigidity of amyloid fibrils. The obtained data indicate that tagging can significantly affect the fibrillogenesis of the target protein.


Assuntos
Amiloide , Glutationa Transferase , Proteína Supressora de Tumor Von Hippel-Lindau , Amiloide/metabolismo , Amiloide/química , Glutationa Transferase/metabolismo , Glutationa Transferase/química , Humanos , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/química , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Microscopia de Força Atômica
2.
Biochem Biophys Res Commun ; 694: 149404, 2024 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-38147698

RESUMO

At the molecular level, aging is often accompanied by dysfunction of stress-induced membrane-less organelles (MLOs) and changes in their physical state (or material properties). In this work, we analyzed the proteins included in the proteome of stress granules (SGs) and P-bodies for their tendency to transform the physical state of these MLOs. Particular attention was paid to the proteins whose gene expression changes during replicative aging. It was shown that the proteome of the studied MLOs consists of intrinsically disordered proteins, 30-40% of which are potentially capable of liquid-liquid phase separation (LLPS). Proteins whose gene expression changes during the transition of human cells to a senescent state make up about 20% of the studied proteomes. There is a statistically significant increase in the number of positively charged proteins in both datasets studied compared to the complete proteomes of these organelles. An increase in the relative content of DNA-, but not RNA-binding proteins, was also found in the SG dataset with senescence-related processes. Among SGs proteins potentially involved in senescent processes, there is an increase in the abundance of potentially amyloidogenic proteins compared to the whole proteome. Proteins common to SGs and P-bodies, potentially involved in processes associated with senescence, form clusters of interacting proteins. The largest cluster is represented by RNA-binding proteins involved in RNA processing and translation regulation. These data indicate that SG proteins, but not proteins of P-bodies, are more likely to transform the physical state of MLOs. Furthermore, these MLOs can participate in processes associated with aging in a coordinated manner.


Assuntos
Corpos de Processamento , Proteoma , Humanos , Proteoma/metabolismo , Grânulos de Estresse , Organelas/metabolismo , Biologia Computacional , Senescência Celular
3.
Biochem Biophys Res Commun ; 693: 149340, 2024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38141525

RESUMO

In addition to the well-known monomeric globular (G-actin) and polymeric fibrillar (F-actin) forms, actin can exist in the so-called inactivated form (I-actin). Hsp70 chaperon, prefoldin, and CCT chaperonin are required to obtain native globular state. In contrast, I-actin is spontaneously formed in the absence of intracellular folding machinery. I-actin can be obtained from G-actin by elimination of divalent ion, incubation in presence of small concentrations of denaturants, and by heat exposure. Since G-actin is a quasi-stationary, thermodynamically unstable form, it can gradually transform into inactivated state in the absence of chelating/denaturating agents or heat exposure, but the transition is much slower. I-actin was shown to associate into oligomers up to the molecular weight of 14-16 G-actin monomers, though the structure of these oligomers remains uncharacterized. This study employs small-angle X-ray scattering to reveal novel insights into the oligomerization process of such spontaneously formed inactivated actin. These oligomers are differentiated from F-actin through comparative analysis, highlighting a unique oligomerization pathway.


Assuntos
Actinas , Dobramento de Proteína , Actinas/metabolismo , Raios X , Proteínas de Choque Térmico HSP70/metabolismo , Quelantes
4.
Cell Mol Life Sci ; 80(9): 269, 2023 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-37634152

RESUMO

The development of aging is associated with the disruption of key cellular processes manifested as well-established hallmarks of aging. Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) have no stable tertiary structure that provide them a power to be configurable hubs in signaling cascades and regulate many processes, potentially including those related to aging. There is a need to clarify the roles of IDPs/IDRs in aging. The dataset of 1702 aging-related proteins was collected from established aging databases and experimental studies. There is a noticeable presence of IDPs/IDRs, accounting for about 36% of the aging-related dataset, which is however less than the disorder content of the whole human proteome (about 40%). A Gene Ontology analysis of the used here aging proteome reveals an abundance of IDPs/IDRs in one-third of aging-associated processes, especially in genome regulation. Signaling pathways associated with aging also contain IDPs/IDRs on different hierarchical levels, revealing the importance of "structure-function continuum" in aging. Protein-protein interaction network analysis showed that IDPs present in different clusters associated with different aging hallmarks. Protein cluster with IDPs enrichment has simultaneously high liquid-liquid phase separation (LLPS) probability, "nuclear" localization and DNA-associated functions, related to aging hallmarks: genomic instability, telomere attrition, epigenetic alterations, and stem cells exhaustion. Intrinsic disorder, LLPS, and aggregation propensity should be considered as features that could be markers of pathogenic proteins. Overall, our analyses indicate that IDPs/IDRs play significant roles in aging-associated processes, particularly in the regulation of DNA functioning. IDP aggregation, which can lead to loss of function and toxicity, could be critically harmful to the cell. A structure-based analysis of aging and the identification of proteins that are particularly susceptible to disturbances can enhance our understanding of the molecular mechanisms of aging and open up new avenues for slowing it down.


Assuntos
Proteínas Intrinsicamente Desordenadas , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteoma , Envelhecimento/genética , Epigenômica , Ontologia Genética
5.
Trends Biochem Sci ; 44(8): 716-728, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31023505

RESUMO

At the turn of this century, cardinal changes took place in the perceptions of the structure and function of proteins, as well as in the organizational principles of membrane-less organelles. As a result, the model of the organization of living matter is changing to one described by highly dynamic biological soft matter positioned at the edge of chaos. Intrinsically disordered proteins (IDPs) and membrane-less organelles are key examples of this new outlook and may represent a critical foundation of life, defining its complexity and the evolution of living things.


Assuntos
Materiais Biocompatíveis/química , Proteínas Intrinsicamente Desordenadas/química , Sequência de Aminoácidos , Transferência Ressonante de Energia de Fluorescência , Organelas/química , Organelas/metabolismo , Transição de Fase , Conformação Proteica , Imagem Individual de Molécula , Temperatura de Transição
6.
Cell Mol Life Sci ; 79(5): 251, 2022 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-35445278

RESUMO

At the turn of the twenty-first century, fundamental changes took place in the understanding of the structure and function of proteins and then in the appreciation of the intracellular space organization. A rather mechanistic model of the organization of living matter, where the function of proteins is determined by their rigid globular structure, and the intracellular processes occur in rigidly determined compartments, was replaced by an idea that highly dynamic and multifunctional "soft matter" lies at the heart of all living things. According this "new view", the most important role in the spatio-temporal organization of the intracellular space is played by liquid-liquid phase transitions of biopolymers. These self-organizing cellular compartments are open dynamic systems existing at the edge of chaos. They are characterized by the exceptional structural and compositional dynamics, and their multicomponent nature and polyfunctionality provide means for the finely tuned regulation of various intracellular processes. Changes in the external conditions can cause a disruption of the biogenesis of these cellular bodies leading to the irreversible aggregation of their constituent proteins, followed by the transition to a gel-like state and the emergence of amyloid fibrils. This work represents a historical overview of changes in our understanding of the intracellular space compartmentalization. It also reflects methodological breakthroughs that led to a change in paradigms in this area of science and discusses modern ideas about the organization of the intracellular space. It is emphasized here that the membrane-less organelles have to combine a certain resistance to the changes in their environment and, at the same time, show high sensitivity to the external signals, which ensures the normal functioning of the cell.


Assuntos
Organelas , Proteínas , Espaço Intracelular/metabolismo , Organelas/metabolismo , Proteínas/metabolismo
7.
Int J Mol Sci ; 24(4)2023 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-36834689

RESUMO

To date, it has been shown that the phenomenon of liquid-liquid phase separation (LLPS) underlies many seemingly completely different cellular processes. This provided a new idea of the spatiotemporal organization of the cell. The new paradigm makes it possible to provide answers to many long-standing, but still unresolved questions facing the researcher. In particular, spatiotemporal regulation of the assembly/disassembly of the cytoskeleton, including the formation of actin filaments, becomes clearer. To date, it has been shown that coacervates of actin-binding proteins that arise during the phase separation of the liquid-liquid type can integrate G-actin and thereby increase its concentration to initiate polymerization. It has also been shown that the activity intensification of actin-binding proteins that control actin polymerization, such as N-WASP and Arp2/3, can be caused by their integration into liquid droplet coacervates formed by signaling proteins on the inner side of the cell membrane.


Assuntos
Actinas , Proteínas dos Microfilamentos , Actinas/metabolismo , Polimerização , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto/metabolismo
8.
Int J Mol Sci ; 24(23)2023 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-38069029

RESUMO

PML bodies are subnuclear protein complexes that play a crucial role in various physiological and pathological cellular processes. One of the general structural proteins of PML bodies is a member of the tripartite motif (TRIM) family-promyelocytic leukemia protein (PML). It is known that PML interacts with over a hundred partners, and the protein itself is represented by several major isoforms, differing in their variable and disordered C-terminal end due to alternative splicing. Despite nearly 30 years of research, the mechanisms underlying PML body formation and the role of PML proteins in this process remain largely unclear. In this review, we examine the literature and highlight recent progress in this field, with a particular focus on understanding the role of individual domains of the PML protein, its post-translational modifications, and polyvalent nonspecific interactions in the formation of PML bodies. Additionally, based on the available literature, we propose a new hypothetical model of PML body formation.


Assuntos
Proteínas Nucleares , Corpos Nucleares da Leucemia Promielocítica , Proteínas Nucleares/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/química , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido
9.
Int J Mol Sci ; 24(13)2023 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-37446185

RESUMO

Radical changes in the idea of the organization of intracellular space that occurred in the early 2010s made it possible to consider the formation and functioning of so-called membrane-less organelles (MLOs) based on a single physical principle: the liquid-liquid phase separation (LLPS) of biopolymers. Weak non-specific inter- and intramolecular interactions of disordered polymers, primarily intrinsically disordered proteins, and RNA, play a central role in the initiation and regulation of these processes. On the other hand, in some cases, the "maturation" of MLOs can be accompanied by a "liquid-gel" phase transition, where other types of interactions can play a significant role in the reorganization of their structure. In this work, we conducted a bioinformatics analysis of the propensity of the proteomes of two membrane-less organelles, formed in response to stress in the same compartment, for spontaneous phase separation and examined their intrinsic disorder predispositions. These MLOs, amyloid bodies (A-bodies) formed in the response to acidosis and heat shock and nuclear stress bodies (nSBs), are characterized by a partially overlapping composition, but show different functional activities and morphologies. We show that the proteomes of these biocondensates are differently enriched in proteins, and many have high potential for spontaneous LLPS that correlates with the different morphology and function of these organelles. The results of these analyses allowed us to evaluate the role of weak interactions in the formation and functioning of these important organelles.


Assuntos
Proteínas Intrinsicamente Desordenadas , Proteoma , Proteoma/metabolismo , Organelas/metabolismo , Nucléolo Celular/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Membrana Nuclear/metabolismo
10.
Int J Mol Sci ; 24(9)2023 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-37175815

RESUMO

At the beginning of the 21st century, it became obvious that radical changes had taken place in the concept of living matter and, in particular, in the concept of the organization of intracellular space. The accumulated data testify to the essential importance of phase transitions of biopolymers (first of all, intrinsically disordered proteins and RNA) in the spatiotemporal organization of the intracellular space. Of particular interest is the stress-induced reorganization of the intracellular space. Examples of organelles formed in response to stress are nuclear A-bodies and nuclear stress bodies. The formation of these organelles is based on liquid-liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs) and non-coding RNA. Despite their overlapping composition and similar mechanism of formation, these organelles have different functional activities and physical properties. In this review, we will focus our attention on these membrane-less organelles (MLOs) and describe their functions, structure, and mechanism of formation.


Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/química , Organelas/metabolismo , Membrana Nuclear/metabolismo , RNA/metabolismo , RNA não Traduzido/metabolismo
11.
Int J Mol Sci ; 25(1)2023 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-38203210

RESUMO

In recent years, the role of liquid-liquid phase separation (LLPS) and intrinsically disordered proteins (IDPs) in cellular molecular processes has received increasing attention from researchers. One such intrinsically disordered protein is TSPYL5, considered both as a marker and a potential therapeutic target for various oncological diseases. However, the role of TSPYL5 in intracellular processes remains unknown, and there is no clarity even in its intracellular localization. In this study, we characterized the intracellular localization and exchange dynamics with intracellular contents of TSPYL5 and its parts, utilizing TSPYL5 fusion proteins with EGFP. Our findings reveal that TSPYL5 can be localized in both the cytoplasm and nucleoplasm, including the nucleolus. The nuclear (nucleolar) localization of TSPYL5 is mediated by the nuclear/nucleolar localization sequences (NLS/NoLS) identified in the N-terminal intrinsically disordered region (4-27 aa), while its cytoplasmic localization is regulated by the ordered NAP-like domain (198-382 aa). Furthermore, our results underscore the significant role of the TSPYL5 N-terminal disordered region (1-198 aa) in the exchange dynamics with the nucleoplasm and its potential ability for phase separation. Bioinformatics analysis of the TSPYL5 interactome indicates its potential function as a histone and ribosomal protein chaperone. Taken together, these findings suggest a significant contribution of liquid-liquid phase separation to the processes involving TSPYL5, providing new insights into the role of this protein in the cell's molecular life.


Assuntos
Histonas , Proteínas Intrinsicamente Desordenadas , Núcleo Celular , Proteínas Ribossômicas , Ribossomos
12.
Int J Mol Sci ; 23(9)2022 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-35563401

RESUMO

Stress is an inevitable part of life. An organism is exposed to multiple stresses and overcomes their negative consequences throughout its entire existence. A correlation was established between life expectancy and resistance to stress, suggesting a relationship between aging and the ability to respond to external adverse effects as well as quickly restore the normal regulation of biological processes. To combat stress, cells developed multiple pro-survival mechanisms, one of them is the assembly of special stress-induced membraneless organelles (MLOs). MLOs are formations that do not possess a lipid membrane but rather form as a result of the "liquid-liquid" phase separation (LLPS) of biopolymers. Stress-responsive MLOs were found in eukaryotes and prokaryotes, they form as a reaction to the acute environmental conditions and are dismantled after its termination. These compartments function to prevent damage to the genetic and protein material of the cell during stress. In this review, we discuss the characteristics of stress-induced MLO-like structures in eukaryotic and prokaryotic cells.


Assuntos
Eucariotos , Organelas , Condensados Biomoleculares , Eucariotos/metabolismo , Organelas/metabolismo , Células Procarióticas/metabolismo , Proteínas/química
13.
Int J Mol Sci ; 23(3)2022 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-35163537

RESUMO

In this work, we performed a comparative study of the formation of PML bodies by full-length PML isoforms and their C-terminal domains in the presence and absence of endogenous PML. Based on the analysis of the distribution of intrinsic disorder predisposition in the amino acid sequences of PML isoforms, regions starting from the amino acid residue 395 (i.e., sequences encoded by exons 4-6) were assigned as the C-terminal domains of these proteins. We demonstrate that each of the full-sized nuclear isoforms of PML is capable of forming nuclear liquid-droplet compartments in the absence of other PML isoforms. These droplets possess dynamic characteristics of the exchange with the nucleoplasm close to those observed in the wild-type cells. Only the C-terminal domains of the PML-II and PML-V isoforms are able to be included in the composition of the endogenous PML bodies, while being partially distributed in the nucleoplasm. The bodies formed by the C-terminal domain of the PML-II isoform are dynamic liquid droplet compartments, regardless of the presence or absence of endogenous PML. The C-terminal domain of PML-V forms dynamic liquid droplet compartments in the knockout cells (PML-/-), but when the C-terminus of the PML-V isoform is inserted into the existing endogenous PML bodies, the molecules of this protein cease to exchange with the nucleoplasm. It was demonstrated that the K490R substitution, which disrupts the PML sumoylation, promotes diffuse distribution of the C-terminal domains of PML-II and PML-V isoforms in endogenous PML knockout HeLa cells, but not in the wild-type cells. These data indicate the ability of the C-terminal domains of the PML-II and PML-V isoforms to form dynamic liquid droplet-like compartments, regardless of the ordered N-terminal RBCC motifs of the PML. This indicates a significant role of the non-specific interactions between the mostly disordered C-terminal domains of PML isoforms for the initiation of liquid-liquid phase separation (LLPS) leading to the formation of PML bodies.


Assuntos
Substituição de Aminoácidos , Corpos Nucleares da Leucemia Promielocítica/metabolismo , Proteína da Leucemia Promielocítica/química , Proteína da Leucemia Promielocítica/metabolismo , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Técnicas de Inativação de Genes , Células HeLa , Humanos , Proteína da Leucemia Promielocítica/genética , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Sumoilação
14.
Photochem Photobiol Sci ; 20(12): 1645-1656, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34796467

RESUMO

Light-oxygen-voltage (LOV) domains are common photosensory modules that found many applications in fluorescence microscopy and optogenetics. Here, we show that the Chloroflexus aggregans LOV domain can bind different flavin species (lumichrome, LC; riboflavin, RF; flavin mononucleotide, FMN; flavin adenine dinucleotide, FAD) during heterologous expression and that its physicochemical properties depend strongly on the nature of the bound flavin. We show that whereas the dissociation constants for different chromophores are similar, the melting temperature of the protein reconstituted with single flavin species varies from ~ 60 °C for LC to ~ 81 °C for FMN, and photobleaching half-times vary almost 100-fold. These observations serve as a caution for future studies of LOV domains in non-native conditions yet raise the possibility of fine-tuning various properties of LOV-based fluorescent probes and optogenetic tools by manipulating the chromophore composition.


Assuntos
Chloroflexus , Oxigênio , Mononucleotídeo de Flavina , Flavina-Adenina Dinucleotídeo , Riboflavina
15.
Biochemistry (Mosc) ; 86(3): 275-289, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33838629

RESUMO

Aging is a prime systemic cause of various age-related diseases, in particular, proteinopathies. In fact, most diseases associated with protein misfolding are sporadic, and their incidence increases with aging. This review examines the process of protein aggregate formation, the toxicity of such aggregates, the organization of cellular systems involved in proteostasis, and the impact of protein aggregates on important cellular processes leading to proteinopathies. We also analyze how manifestations of aging (mitochondrial dysfunction, dysfunction of signaling systems, changes in the genome and epigenome) facilitate pathogenesis of various proteinopathies either directly, by increasing the propensity of key proteins for aggregation, or indirectly, through dysregulation of stress responses. Such analysis might help in outlining approaches for treating proteinopathies and extending healthy longevity.


Assuntos
Envelhecimento , Agregação Patológica de Proteínas , Deficiências na Proteostase/metabolismo , Animais , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Proteostase , Deficiências na Proteostase/etiologia , Deficiências na Proteostase/fisiopatologia
16.
Int J Mol Sci ; 22(20)2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34681772

RESUMO

The fluorescent dye BADAN (6-bromoacetyl-2-dimetylaminonaphtalene) is widely used in various fields of life sciences, however, the photophysical properties of BADAN are not fully understood. The study of the spectral properties of BADAN attached to a number of mutant forms of GGBP, as well as changes in its spectral characteristics during structural changes in proteins, allowed to shed light on the photophysical properties of BADAN. It was shown that spectral properties of BADAN are determined by at least one non-fluorescent and two fluorescent isomers with overlapping absorbing bands. It was found that BADAN fluorescence is determined by the unsolvated "PICT" (planar intramolecular charge transfer state) and solvated "TICT" (twisted intramolecular charge transfer state) excited states. While "TICT" state can be formed both as a result of the "PICT" state solvation and as a result of light absorption by the solvated ground state of the dye. BADAN fluorescence linked to GGBP/H152C apoform is quenched by Trp 183, but this effect is inhibited by glucose intercalation. New details of the changes in the spectral characteristics of BADAN during the unfolding of the protein apo and holoforms have been obtained.


Assuntos
2-Naftilamina/análogos & derivados , Proteínas de Escherichia coli/química , Proteínas de Transporte de Monossacarídeos/química , 2-Naftilamina/química , 2-Naftilamina/farmacologia , Substituição de Aminoácidos , Escherichia coli , Proteínas de Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fluorescência , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Proteínas de Transporte de Monossacarídeos/efeitos dos fármacos , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Mutação de Sentido Incorreto , Conformação Proteica/efeitos dos fármacos , Espectrometria de Fluorescência/métodos , Relação Estrutura-Atividade
17.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072343

RESUMO

In this work, we put forward a hypothesis about the decisive role of multivalent nonspecific interactions in the early stages of PML body formation. Our analysis of the PML isoform sequences showed that some of the PML isoforms, primarily PML-II, are prone to phase separation due to their polyampholytic properties and the disordered structure of their C-terminal domains. The similarity of the charge properties of the C-terminal domains of PML-II and PML-VI isoforms made it possible for the first time to detect migration of PML-VI from PML bodies to the periphery of the cell nucleus, similar to the migration of PML-II isoforms. We found a population of "small" (area less than 1 µm2) spherical PML bodies with high dynamics of PML isoforms exchange with nucleoplasm and a low fraction of immobilized proteins, which indicates their liquid state properties. Such structures can act as "seeds" of functionally active PML bodies, providing the necessary concentration of PML isoforms for the formation of intermolecular disulfide bonds between PML monomers. FRAP analysis of larger bodies of toroidal topology showed the existence of an insoluble scaffold in their structure. The hypothesis about the role of nonspecific multiple weak interactions in the formation of PML bodies is further supported by the change in the composition of the scaffold proteins of PML bodies, but not their solidification, under conditions of induction of dimerization of PML isoforms under oxidative stress. Using the colocalization of ALT-associated PML bodies (APBs) with TRF1, we identified APBs and showed the difference in the dynamic properties of APBs and canonical PML bodies.


Assuntos
Corpos de Inclusão Intranuclear/metabolismo , Proteína da Leucemia Promielocítica/metabolismo , Telômero/genética , Telômero/metabolismo , Sequência de Aminoácidos , Biomarcadores , Núcleo Celular/metabolismo , Imunofluorescência , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Imagem Molecular , Estresse Oxidativo , Proteína da Leucemia Promielocítica/química , Proteína da Leucemia Promielocítica/genética , Ligação Proteica , Isoformas de Proteínas , Transporte Proteico , Homeostase do Telômero
18.
Cell Mol Life Sci ; 76(22): 4461-4492, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31428838

RESUMO

GPCR-G protein signaling system recognizes a multitude of extracellular ligands and triggers a variety of intracellular signaling cascades in response. In humans, this system includes more than 800 various GPCRs and a large set of heterotrimeric G proteins. Complexity of this system goes far beyond a multitude of pair-wise ligand-GPCR and GPCR-G protein interactions. In fact, one GPCR can recognize more than one extracellular signal and interact with more than one G protein. Furthermore, one ligand can activate more than one GPCR, and multiple GPCRs can couple to the same G protein. This defines an intricate multifunctionality of this important signaling system. Here, we show that the multifunctionality of GPCR-G protein system represents an illustrative example of the protein structure-function continuum, where structures of the involved proteins represent a complex mosaic of differently folded regions (foldons, non-foldons, unfoldons, semi-foldons, and inducible foldons). The functionality of resulting highly dynamic conformational ensembles is fine-tuned by various post-translational modifications and alternative splicing, and such ensembles can undergo dramatic changes at interaction with their specific partners. In other words, GPCRs and G proteins exist as sets of conformational/basic, inducible/modified, and functioning proteoforms characterized by a broad spectrum of structural features and possessing various functional potentials.


Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Animais , Humanos , Ligantes , Processamento de Proteína Pós-Traducional/fisiologia
20.
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
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