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1.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360917

RESUMO

Although once perceived as inert structures that merely serve for lipid storage, lipid droplets (LDs) have proven to be the dynamic organelles that hold many cellular functions. The LDs' basic structure of a hydrophobic core consisting of neutral lipids and enclosed in a phospholipid monolayer allows for quick lipid accessibility for intracellular energy and membrane production. Whereas formed at the peripheral and perinuclear endoplasmic reticulum, LDs are degraded either in the cytosol by lipolysis or in the vacuoles/lysosomes by autophagy. Autophagy is a regulated breakdown of dysfunctional, damaged, or surplus cellular components. The selective autophagy of LDs is called lipophagy. Here, we review LDs and their degradation by lipophagy in yeast, which proceeds via the micrometer-scale raft-like lipid domains in the vacuolar membrane. These vacuolar microdomains form during nutrient deprivation and facilitate internalization of LDs via the vacuolar membrane invagination and scission. The resultant intra-vacuolar autophagic bodies with LDs inside are broken down by vacuolar lipases and proteases. This type of lipophagy is called microlipophagy as it resembles microautophagy, the type of autophagy when substrates are sequestered right at the surface of a lytic compartment. Yeast microlipophagy via the raft-like vacuolar microdomains is a great model system to study the role of lipid domains in microautophagic pathways.


Assuntos
Autofagia/fisiologia , Gotículas Lipídicas/metabolismo , Microdomínios da Membrana/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Homeostase/fisiologia , Lipólise/fisiologia , Lisossomos/metabolismo , Fosfolipídeos/metabolismo
2.
Nat Commun ; 12(1): 4905, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385458

RESUMO

α-ketoglutarate (KG), also referred to as 2-oxoglutarate, is a key intermediate of cellular metabolism with pleiotropic functions. Cell-permeable esterified analogs are widely used to study how KG fuels bioenergetic and amino acid metabolism and DNA, RNA, and protein hydroxylation reactions, as cellular membranes are thought to be impermeable to KG. Here we show that esterified KG analogs rapidly hydrolyze in aqueous media, yielding KG that, in contrast to prevailing assumptions, imports into many cell lines. Esterified KG analogs exhibit spurious KG-independent effects on cellular metabolism, including extracellular acidification, arising from rapid hydrolysis and de-protonation of α-ketoesters, and significant analog-specific inhibitory effects on glycolysis or mitochondrial respiration. We observe that imported KG decarboxylates to succinate in the cytosol and contributes minimally to mitochondrial metabolism in many cell lines cultured in normal conditions. These findings demonstrate that nuclear and cytosolic KG-dependent reactions may derive KG from functionally distinct subcellular pools and sources.


Assuntos
Aminoácidos/metabolismo , Metabolismo Energético , Ésteres/metabolismo , Ácidos Cetoglutáricos/metabolismo , Mitocôndrias/metabolismo , Ácido Succínico/metabolismo , Animais , Linhagem Celular Tumoral , Citosol/metabolismo , Ésteres/química , Glicólise , Células HEK293 , Humanos , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Ácidos Cetoglutáricos/química , Camundongos , Consumo de Oxigênio , Células RAW 264.7
3.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445484

RESUMO

In response to diverse pathogenic and danger signals, the cytosolic activation of the NLRP3 (NOD-, LRR-, and pyrin domain-containing (3)) inflammasome complex is a critical event in the maturation and release of some inflammatory cytokines in the state of an inflammatory response. After activation of the NLRP3 inflammasome, a series of cellular events occurs, including caspase 1-mediated proteolytic cleavage and maturation of the IL-1ß and IL-18, followed by pyroptotic cell death. Therefore, the NLRP3 inflammasome has become a prime target for the resolution of many inflammatory disorders. Since NLRP3 inflammasome activation can be triggered by a wide range of stimuli and the activation process occurs in a complex, it is difficult to target the NLRP3 inflammasome. During the activation process, various post-translational modifications (PTM) of the NLRP3 protein are required to form a complex with other components. The regulation of ubiquitination and deubiquitination of NLRP3 has emerged as a potential therapeutic target for NLRP3 inflammasome-associated inflammatory disorders. In this review, we discuss the ubiquitination and deubiquitination system for NLRP3 inflammasome activation and the inhibitors that can be used as potential therapeutic agents to modulate the activation of the NLRP3 inflammasome.


Assuntos
Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Citosol/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Terapia de Alvo Molecular , Ubiquitinação/efeitos dos fármacos
4.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445509

RESUMO

Ischemia-like conditions reflect almost the entire spectrum of events that occur during cerebral ischemia, including the induction of oxidative stress, Ca2+ overload, glutamate excitotoxicity, and activation of necrosis and apoptosis in brain cells. Mechanisms for the protective effects of the antioxidant enzyme peroxiredoxin-6 (Prx-6) on hippocampal cells during oxygen-glucose deprivation/reoxygenation (OGD/R) were investigated. Using the methods of fluorescence microscopy, inhibitory analysis, vitality tests and PCR, it was shown that 24-h incubation of mixed hippocampal cell cultures with Prx-6 does not affect the generation of a reversible phase of a OGD-induced rise in Ca2+ ions in cytosol ([Ca2+]i), but inhibits a global increase in [Ca2+]i in astrocytes completely and in neurons by 70%. In addition, after 40 min of OGD, cell necrosis is suppressed, especially in the astrocyte population. This effect is associated with the complex action of Prx-6 on neuroglial networks. As an antioxidant, Prx-6 has a more pronounced and astrocyte-directed effect, compared to the exogenous antioxidant vitamin E (Vit E). Prx-6 inhibits ROS production in mitochondria by increasing the antioxidant capacity of cells and altering the expression of genes encoding redox status proteins. Due to the close bond between [Ca2+]i and intracellular ROS, this effect of Prx-6 is one of its protective mechanisms. Moreover, Prx-6 effectively suppresses not only necrosis, but also apoptosis during OGD and reoxygenation. Incubation with Prx-6 leads to activation of the basic expression of genes encoding protective kinases-PI3K, CaMKII, PKC, anti-apoptotic proteins-Stat3 and Bcl-2, while inhibiting the expression of signaling kinases and factors involved in apoptosis activation-Ikk, Src, NF-κb, Caspase-3, p53, Fas, etc. This effect on the basic expression of the genome leads to the cell preconditions, which is expressed in the inhibition of caspase-3 during OGD/reoxygenation. A significant effect of Prx-6 is directed on suppression of the level of pro-inflammatory cytokine IL-1ß and factor TNFα, as well as genes encoding NMDA- and kainate receptor subunits, which was established for the first time for this antioxidant enzyme. The protective effect of Prx-6 is due to its antioxidant properties, since mutant Prx-6 (mutPrx-6, Prx6-C47S) leads to polar opposite effects, contributing to oxidative stress, activation of apoptosis and cell death through receptor action on TLR4.


Assuntos
Astrócitos/citologia , Hipocampo/citologia , Peroxirredoxina VI/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Cálcio/metabolismo , Células Cultivadas , Citosol/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Interleucina-1beta/metabolismo , Microscopia de Fluorescência , Peroxirredoxina VI/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
5.
Viruses ; 13(7)2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34372555

RESUMO

Viroplasms are cytoplasmic, membraneless structures assembled in rotavirus (RV)-infected cells, which are intricately involved in viral replication. Two virus-encoded, non-structural proteins, NSP2 and NSP5, are the main drivers of viroplasm formation. The structures (as far as is known) and functions of these proteins are described. Recent studies using plasmid-only-based reverse genetics have significantly contributed to elucidation of the crucial roles of these proteins in RV replication. Thus, it has been recognized that viroplasms resemble liquid-like protein-RNA condensates that may be formed via liquid-liquid phase separation (LLPS) of NSP2 and NSP5 at the early stages of infection. Interactions between the RNA chaperone NSP2 and the multivalent, intrinsically disordered protein NSP5 result in their condensation (protein droplet formation), which plays a central role in viroplasm assembly. These droplets may provide a unique molecular environment for the establishment of inter-molecular contacts between the RV (+)ssRNA transcripts, followed by their assortment and equimolar packaging. Future efforts to improve our understanding of RV replication and genome assortment in viroplasms should focus on their complex molecular composition, which changes dynamically throughout the RV replication cycle, to support distinct stages of virion assembly.


Assuntos
Rotavirus/genética , Rotavirus/metabolismo , Compartimentos de Replicação Viral/metabolismo , Animais , Proteínas do Capsídeo/genética , Citoplasma/virologia , Citosol/metabolismo , Humanos , Fosforilação , Proteínas de Ligação a RNA/metabolismo , Infecções por Rotavirus/virologia , Proteínas não Estruturais Virais/metabolismo , Compartimentos de Replicação Viral/fisiologia , Montagem de Vírus , Replicação Viral/genética
6.
Nat Commun ; 12(1): 4932, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389733

RESUMO

BAX is a pro-apoptotic member of the BCL-2 family, which regulates the balance between cellular life and death. During homeostasis, BAX predominantly resides in the cytosol as a latent monomer but, in response to stress, transforms into an oligomeric protein that permeabilizes the mitochondria, leading to apoptosis. Because renegade BAX activation poses a grave risk to the cell, the architecture of BAX must ensure monomeric stability yet enable conformational change upon stress signaling. The specific structural features that afford both stability and dynamic flexibility remain ill-defined and represent a critical control point of BAX regulation. We identify a nexus of interactions involving four residues of the BAX core α5 helix that are individually essential to maintaining the structure and latency of monomeric BAX and are collectively required for dimeric assembly. The dual yet distinct roles of these residues reveals the intricacy of BAX conformational regulation and opportunities for therapeutic modulation.


Assuntos
Aminoácidos/genética , Apoptose/genética , Mutação , Transdução de Sinais/genética , Proteína X Associada a bcl-2/genética , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Sítios de Ligação/genética , Células Cultivadas , Citosol/metabolismo , Humanos , Camundongos Knockout , Mitocôndrias/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo
7.
Nat Commun ; 12(1): 4855, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381044

RESUMO

The vertebrate brain consists of diverse neuronal types, classified by distinct anatomy and function, along with divergent transcriptomes and proteomes. Defining the cell-type specific neuroproteomes is important for understanding the development and functional organization of neural circuits. This task remains challenging in complex tissue, due to suboptimal protein isolation techniques that often result in loss of cell-type specific information and incomplete capture of subcellular compartments. Here, we develop a genetically targeted proximity labeling approach to identify cell-type specific subcellular proteomes in the mouse brain, confirmed by imaging, electron microscopy, and mass spectrometry. We virally express subcellular-localized APEX2 to map the proteome of direct and indirect pathway spiny projection neurons in the striatum. The workflow provides sufficient depth to uncover changes in the proteome of striatal neurons following chemogenetic activation of Gαq-coupled signaling cascades. This method enables flexible, cell-type specific quantitative profiling of subcellular proteome snapshots in the mouse brain.


Assuntos
Ascorbato Peroxidases/metabolismo , Núcleo Celular/metabolismo , Corpo Estriado/metabolismo , Proteoma/metabolismo , Animais , Ascorbato Peroxidases/genética , Corpo Estriado/citologia , Citosol/metabolismo , Espectrometria de Massas , Camundongos , Vias Neurais , Neurônios/citologia , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Coloração e Rotulagem , Fluxo de Trabalho
8.
Nat Commun ; 12(1): 4284, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257281

RESUMO

The translocase of the outer mitochondrial membrane TOM constitutes the organellar entry gate for nearly all precursor proteins synthesized on cytosolic ribosomes. Thus, TOM presents the ideal target to adjust the mitochondrial proteome upon changing cellular demands. Here, we identify that the import receptor TOM70 is targeted by the kinase DYRK1A and that this modification plays a critical role in the activation of the carrier import pathway. Phosphorylation of TOM70Ser91 by DYRK1A stimulates interaction of TOM70 with the core TOM translocase. This enables transfer of receptor-bound precursors to the translocation pore and initiates their import. Consequently, loss of TOM70Ser91 phosphorylation results in a strong decrease in import capacity of metabolite carriers. Inhibition of DYRK1A impairs mitochondrial structure and function and elicits a protective transcriptional response to maintain a functional import machinery. The DYRK1A-TOM70 axis will enable insights into disease mechanisms caused by dysfunctional DYRK1A, including autism spectrum disorder, microcephaly and Down syndrome.


Assuntos
Transtorno do Espectro Autista/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transtorno do Espectro Autista/genética , Citosol/metabolismo , Síndrome de Down/genética , Síndrome de Down/metabolismo , Humanos , Microcefalia/genética , Microcefalia/metabolismo , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética
9.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209978

RESUMO

Mitochondria are regarded as the metabolic centers of cells and are integral in many other cell processes, including the immune response. Each mitochondrion contains numerous copies of mitochondrial DNA (mtDNA), a small, circular, and bacterial-like DNA. In response to cellular damage or stress, mtDNA can be released from the mitochondrion and trigger immune and inflammatory responses. mtDNA release into the cytosol or bloodstream can occur as a response to hypoxia, sepsis, traumatic injury, excitatory cytotoxicity, or drastic mitochondrial membrane potential changes, some of which are hallmarks of neurodegenerative and mood disorders. Released mtDNA can mediate inflammatory responses observed in many neurological and mood disorders by driving the expression of inflammatory cytokines and the interferon response system. The current understanding of the role of mtDNA release in affective mood disorders and neurodegenerative diseases will be discussed.


Assuntos
DNA Mitocondrial/genética , Mitocôndrias/genética , Doenças Neurodegenerativas/genética , Animais , Citosol/metabolismo , Humanos , Mutação , Doenças Neurodegenerativas/imunologia
10.
Int J Mol Sci ; 22(14)2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34298920

RESUMO

Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application in living cells, and we were able to distinguish GFP, diGFP, as well as ligand-induced dimerization of FKBP12 (FK506 binding protein 12)-GFP. While other analysis methods have only sporadically been used to study dimerization in living cells and may be prone to errors, this paper provides a robust approach for the investigation of any cytosolic protein using single-color fluorescence fluctuation spectroscopy.


Assuntos
Multimerização Proteica/fisiologia , Proteínas/metabolismo , Células Cultivadas , Citosol/metabolismo , Dictyostelium/metabolismo , Difusão , Dimerização , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Ligantes , Fótons , Espectrometria de Fluorescência/métodos
11.
Nat Commun ; 12(1): 4514, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301929

RESUMO

Regulation of IP3 receptors (IP3Rs) by IP3 and Ca2+ allows regenerative Ca2+ signals, the smallest being Ca2+ puffs, which arise from coordinated openings of a few clustered IP3Rs. Cells express thousands of mostly mobile IP3Rs, yet Ca2+ puffs occur at a few immobile IP3R clusters. By imaging cells with endogenous IP3Rs tagged with EGFP, we show that KRas-induced actin-interacting protein (KRAP) tethers IP3Rs to actin beneath the plasma membrane. Loss of KRAP abolishes Ca2+ puffs and the global increases in cytosolic Ca2+ concentration evoked by more intense stimulation. Over-expressing KRAP immobilizes additional IP3R clusters and results in more Ca2+ puffs and larger global Ca2+ signals. Endogenous KRAP determines which IP3Rs will respond: it tethers IP3R clusters to actin alongside sites where store-operated Ca2+ entry occurs, licenses IP3Rs to evoke Ca2+ puffs and global cytosolic Ca2+ signals, implicates the actin cytoskeleton in IP3R regulation and may allow local activation of Ca2+ entry.


Assuntos
Actinas/metabolismo , Cálcio/metabolismo , Citosol/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Células HEK293 , Células HeLa , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Proteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Microscopia de Fluorescência , Ligação Proteica , Interferência de RNA
12.
Methods Mol Biol ; 2342: 737-763, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34272715

RESUMO

In the first edition of this book, we presented the basics of explicitly incorporating the lipid biochemistry into a confluent cell monolayer transport model and the novel findings of this model up to 2013, including the use of global optimization to fit the elementary rate constants and the efflux active P-glycoprotein (P-gp) membrane concentrations for the transport of four P-gp substrates across MDCKII-hMDR1-NKI confluent cell monolayers. This chapter is an update on that model, which has been focused primarily on discovering how microvilli morphology regulates the efflux active P-gp and the existence of, as yet, unidentified uptake transporters of P-gp substrates in all of the commonly used P-gp expressing cell lines used in the pharmaceutical industry, thereby adding new players to DDI predictions and IVIVE. The structural mass action kinetic model uses the general mass action reactions for P-gp binding and efflux, with the membrane structural parameters for the confluent cell monolayer to predict drug transport over time. Binding of drug to P-gp occurs within the cytosolic monolayer of the apical membrane, according to (a) the molar partition coefficient of the drug to the cytosolic monolayer and (b) the association rate constant, k1 (M-1 s-1), of the drug from the basolateral or apical outer monolayers into the P-gp binding site. Release of substrate from P-gp back into the cytosolic monolayer occurs with a dissociation rate constant kr (s-1) or, much less frequently, into the apical aqueous chamber with an efflux rate constant k2 (s-1). The model fits the efflux active P-gp concentration, T(0), i.e., the P-gp whose effluxed drug actually reaches the apical aqueous chamber, as opposed to the majority of P-gp whose effluxed drug is reabsorbed back into the same or neighboring microvilli prior to reaching the apical aqueous chamber. Efflux active P-gp largely resides near the tips of the microvilli. We have shown using kinetics and structured illumination microscopy that: (a) efflux active P-gp is controlled by microvilli morphology; (b) there are apical (AT) and basolateral (BT) uptake transporters for P-gp substrates in most, if not all, P-gp expressing cell lines used in the pharmaceutical industry, which exist, but which remain unidentified; (c) the lab-to-lab variability in P-gp IC50 values observed in the P-gp IC50 initiative was due to the conflated inhibition of P-gp and the basolateral digoxin uptake transporters by all 15 P-gp substrates tested in that study; (d) even the IC50 values for P-gp inhibition alone do not obey the Cheng-Prusoff relationship; (e) the fitted elementary rate constants and the molecular dissociation constant Ki for this kinetic model are system independent; and (f) the time dependence of product formation for these confluent cell monolayers is correlated with the P-gp Vmax/Km, when defined by its fitted elementary rate constants and uptake transporter clearances, without any steady-state assumptions.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Técnicas de Cultura de Células/métodos , Preparações Farmacêuticas/metabolismo , Animais , Sítios de Ligação , Transporte Biológico , Células CACO-2 , Células Cultivadas , Citosol/metabolismo , Humanos , Cinética , Microvilosidades/metabolismo , Modelos Teóricos
13.
Commun Biol ; 4(1): 843, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234268

RESUMO

Age-related changes in cellular metabolism can affect brain homeostasis, creating conditions that are permissive to the onset and progression of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Although the roles of metabolites have been extensively studied with regard to cellular signaling pathways, their effects on protein aggregation remain relatively unexplored. By computationally analysing the Human Metabolome Database, we identified two endogenous metabolites, carnosine and kynurenic acid, that inhibit the aggregation of the amyloid beta peptide (Aß) and rescue a C. elegans model of Alzheimer's disease. We found that these metabolites act by triggering a cytosolic unfolded protein response through the transcription factor HSF-1 and downstream chaperones HSP40/J-proteins DNJ-12 and DNJ-19. These results help rationalise previous observations regarding the possible anti-ageing benefits of these metabolites by providing a mechanism for their action. Taken together, our findings provide a link between metabolite homeostasis and protein homeostasis, which could inspire preventative interventions against neurodegenerative disorders.


Assuntos
Doença de Alzheimer/metabolismo , Caenorhabditis elegans/metabolismo , Carnosina/metabolismo , Modelos Animais de Doenças , Ácido Cinurênico/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Doença de Alzheimer/prevenção & controle , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Carnosina/farmacologia , Citosol/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Ácido Cinurênico/farmacologia , Agregados Proteicos , Agregação Patológica de Proteínas/prevenção & controle , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos
14.
Nano Lett ; 21(14): 6022-6030, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34227381

RESUMO

Despite the well-recognized clinical success of therapeutic proteins, especially antibodies, their inability to penetrate the cell membrane restricts them to secretory extracellular or membrane-associated targets. Developing a direct cytosolic protein delivery system would offer unique opportunities for intracellular target-related therapeutic proteins. Here, we generated a supercharged polypeptide (SCP) with high cellular uptake efficiency, endosomal escape ability, and good biosafety and developed an SCP with an unnatural amino acid containing the phenylboronic acid (PBA) group, called PBA-SCP. PBA-SCP is capable of potently delivering proteins with various isoelectric points and molecular sizes into the cytosol of living cells without affecting their bioactivities. Importantly, cytosolically delivered antibodies remain functional and are capable of targeting, labeling, and manipulating diverse intracellular antigens. This study demonstrates an efficient and versatile intracellular protein delivery platform, especially for antibodies, and provides new possibilities for expanding protein-based therapeutics to intracellular "undruggable" targets.


Assuntos
Peptídeos , Proteínas , Transporte Biológico , Citosol/metabolismo , Endossomos/metabolismo , Peptídeos/metabolismo
15.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202520

RESUMO

Trypanosoma brucei (Tb) harbours twelve Hsp70 chaperones. Of these, four are predicted to reside in the parasite cytosol. TbHsp70.c is predicted to be cytosolic and upregulated upon heat stress and is an ATPase that exhibits holdase chaperone function. Cytosol-localized Tbj2 stimulates the ATPase activity of TbHsp70.c. In the current study, immunofluorescence confirmed that TbHsp70.c is both a cytosolic and a nuclear protein. Furthermore, in silico analysis was used to elucidate an atypical linker and hydrophobic pocket. Tellingly, TbHsp70.c lacks the EEVD and GGMP motifs, both of which are implicated in substrate selectivity and co-chaperone binding in canonical Hsp70s. Far western analysis revealed that TbSTi1 interacts directly with TbHsp70 and TbHsp70.4, but does not bind TbHsp70.c. We further investigated the effect of quercetin and methylene blue on the Tbj2-driven ATPase activity of TbHsp70.c. We established that quercetin inhibited, whilst methylene blue enhanced, the Tbj2-stimulated ATPase activity of TbHsp70.c. Furthermore, these inhibitors were lethal to parasites. Lastly, we used molecular docking to show that quercetin and methylene blue may bind the nucleotide binding pocket of TbHsp70.c. Our findings suggest that small molecule inhibitors that target TbHsp70.c could be developed to serve as possible drug candidates against T. brucei.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/classificação , Trypanosoma brucei brucei/fisiologia , Sequência de Aminoácidos , Sítios de Ligação , Núcleo Celular/metabolismo , Citosol/metabolismo , Imunofluorescência , Proteínas de Choque Térmico HSP70/química , Azul de Metileno/química , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Ligação Proteica , Conformação Proteica , Transporte Proteico , Proteínas de Protozoários/química , Quercetina/química , Coloração e Rotulagem , Relação Estrutura-Atividade
16.
Molecules ; 26(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208178

RESUMO

The heat shock protein (HSP) 70 is considered the main hallmark in preclinical studies to stain the peri-infarct region defined area penumbra in preclinical models of brain ischemia. This protein is also considered as a potential disease modifier, which may improve the outcome of ischemic damage. In fact, the molecule HSP70 acts as a chaperonine being able to impact at several level the homeostasis of neurons. Despite being used routinely to stain area penumbra in light microscopy, the subcellular placement of this protein within area penumbra neurons, to our knowledge, remains undefined. This is key mostly when considering studies aimed at deciphering the functional role of this protein as a determinant of neuronal survival. The general subcellular placement of HSP70 was grossly reported in studies using confocal microscopy, although no direct visualization of this molecule at electron microscopy was carried out. The present study aims to provide a direct evidence of HSP70 within various subcellular compartments. In detail, by using ultrastructural morphometry to quantify HSP70 stoichiometrically detected by immuno-gold within specific organelles we could compare the compartmentalization of the molecule within area penumbra compared with control brain areas. The study indicates that two cell compartments in control conditions own a high density of HSP70, cytosolic vacuoles and mitochondria. In these organelles, HSP70 is present in amount exceeding several-fold the presence in the cytosol. Remarkably, within area penumbra a loss of such a specific polarization is documented. This leads to the depletion of HSP70 from mitochondria and mostly cell vacuoles. Such an effect is expected to lead to significant variations in the ability of HSP70 to exert its physiological roles. The present findings, beyond defining the neuronal compartmentalization of HSP70 within area penumbra may lead to a better comprehension of its beneficial/detrimental role in promoting neuronal survival.


Assuntos
Isquemia Encefálica/metabolismo , Citosol/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Vacúolos/metabolismo , Animais , Isquemia Encefálica/patologia , Morte Celular/fisiologia , Citosol/patologia , Modelos Animais de Doenças , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia Eletrônica de Varredura , Mitocôndrias/patologia , Neurônios/patologia , Vacúolos/patologia
17.
Nat Commun ; 12(1): 3721, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140497

RESUMO

Cytosolic transport is an essential requirement but a major obstacle to efficient delivery of therapeutic peptides, proteins and nucleic acids. Current understanding of cytosolic delivery mechanisms remains limited due to a significant number of conflicting reports, which are compounded by low sensitivity and indirect assays. To resolve this, we develop a highly sensitive Split Luciferase Endosomal Escape Quantification (SLEEQ) assay to probe mechanisms of cytosolic delivery. We apply SLEEQ to evaluate the cytosolic delivery of a range of widely studied cell-penetrating peptides (CPPs) fused to a model protein. We demonstrate that positively charged CPPs enhance cytosolic delivery as a result of increased non-specific cell membrane association, rather than increased endosomal escape efficiency. These findings transform our current understanding of how CPPs increase cytosolic delivery. SLEEQ is a powerful tool that addresses fundamental questions in intracellular drug delivery and will significantly improve the way materials are engineered to increase therapeutic delivery to the cytosol.


Assuntos
Membrana Celular/metabolismo , Peptídeos Penetradores de Células/metabolismo , Citosol/metabolismo , Endossomos/metabolismo , Medições Luminescentes/métodos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Humanos , Luciferases/química , Espectrometria de Massas , Proteínas Recombinantes , Sensibilidade e Especificidade
18.
Viruses ; 13(6)2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34064125

RESUMO

To initiate infection, a virus enters a host cell typically via receptor-dependent endocytosis. It then penetrates a subcellular membrane, reaching a destination that supports transcription, translation, and replication of the viral genome. These steps lead to assembly and morphogenesis of the new viral progeny. The mature virus finally exits the host cell to begin the next infection cycle. Strikingly, viruses hijack host molecular chaperones to accomplish these distinct entry steps. Here we highlight how DNA viruses, including polyomavirus and the human papillomavirus, exploit soluble and membrane-associated chaperones to enter a cell, penetrating and escaping an intracellular membrane en route for infection. We also describe the mechanism by which RNA viruses-including flavivirus and coronavirus-co-opt cytosolic and organelle-selective chaperones to promote viral endocytosis, protein biosynthesis, replication, and assembly. These examples underscore the importance of host chaperones during virus infection, potentially revealing novel antiviral strategies to combat virus-induced diseases.


Assuntos
Vírus de DNA/fisiologia , Chaperonas Moleculares/metabolismo , Vírus de RNA/fisiologia , Citosol/metabolismo , Vírus de DNA/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Endossomos/metabolismo , Endossomos/virologia , Interações Hospedeiro-Patógeno , Membranas Intracelulares/metabolismo , Vírus de RNA/metabolismo , Internalização do Vírus , Replicação Viral
19.
FASEB J ; 35(7): e21712, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34110637

RESUMO

Palmitic acid (PA) is a saturated fatty acid whose high consumption has been largely associated with the development of different metabolic alterations, such as insulin resistance, metabolic syndrome, and type 2 diabetes. Particularly in the brain, insulin signaling disruption has been linked to cognitive decline and is considered a risk factor for Alzheimer's disease. Cumulative evidence has demonstrated the participation of PA in the molecular cascade underlying cellular insulin resistance in peripheral tissues, but its role in the development of neuronal insulin resistance and the mechanisms involved are not fully understood. It has generally been accepted that the brain does not utilize fatty acids as a primary energy source, but recent evidence shows that neurons possess the machinery for fatty acid ß-oxidation. However, it is still unclear under what conditions neurons use fatty acids as energy substrates and the implications of their oxidative metabolism in modifying insulin-stimulated effects. In the present work, we have found that neurons differentiated from human neuroblastoma MSN exposed to high but nontoxic concentrations of PA generate ATP through mitochondrial metabolism, which is associated with an increase in the cytosolic Ca2+ and diminished insulin signaling in neurons. These findings reveal a novel mechanism by which saturated fatty acids produce Ca2+ entry and insulin resistance that may play a causal role in increasing neuronal vulnerability associated with metabolic diseases.


Assuntos
Cálcio/metabolismo , Metabolismo Energético/efeitos dos fármacos , Resistência à Insulina/fisiologia , Neurônios/efeitos dos fármacos , Ácido Palmítico/farmacologia , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Citosol/efeitos dos fármacos , Citosol/metabolismo , Ácidos Graxos/farmacologia , Humanos , Insulina/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neuroblastoma/metabolismo , Neurônios/metabolismo , Transdução de Sinais/efeitos dos fármacos
20.
J Exp Bot ; 72(15): 5312-5335, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34077536

RESUMO

Calcium (Ca2+)-dependent signalling plays a well-characterized role in the response to different environmental stimuli, in both plant and animal cells. In the model organism for green algae, Chlamydomonas reinhardtii, Ca2+ signals were reported to have a crucial role in different physiological processes, such as stress responses, photosynthesis, and flagella functions. Recent reports identified the underlying components of the Ca2+ signalling machinery at the level of specific subcellular compartments and reported in vivo imaging of cytosolic Ca2+ concentration in response to environmental stimuli. The characterization of these Ca2+-related mechanisms and proteins in C. reinhardtii is providing knowledge on how microalgae can perceive and respond to environmental stimuli, but also on how this Ca2+ signalling machinery has evolved. Here, we review current knowledge on the cellular mechanisms underlying the generation, shaping, and decoding of Ca2+ signals in C. reinhardtii, providing an overview of the known and possible molecular players involved in the Ca2+ signalling of its different subcellular compartments. The advanced toolkits recently developed to measure time-resolved Ca2+ signalling in living C. reinhardtii cells are also discussed, suggesting how they can improve the study of the role of Ca2+ signals in the cellular response of microalgae to environmental stimuli.


Assuntos
Chlamydomonas reinhardtii , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Chlamydomonas reinhardtii/metabolismo , Citosol/metabolismo , Flagelos/metabolismo
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