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Stress granules (SGs) are cytoplasmic messenger ribonucleoprotein granules transiently formed in stressed mammalian cells. Although SG components have been well characterized, detailed insights into the molecular behavior inside SGs remain unresolved. We investigated nanoscale dynamics and localization of endogenous mRNAs in SGs combining single mRNA tracking and super-resolution localization microscopy. First, we developed a methodology for tracking single mRNAs within SGs, revealing that although mRNAs in SGs are mainly stationary (â¼40%), they also move in a confined (â¼25%) or freely diffusing (â¼35%) manner. Second, the super-resolution localization microscopy showed that the mRNAs in SGs are heterogeneously distributed and partially form high-density clusters. Third, we simultaneously performed single mRNA tracking and super-resolution microscopy in SGs, demonstrating that single mRNA trajectories are mainly found around high-density clusters. Finally, a quantitative analysis of mRNA localization and dynamics during stress removal was conducted using live super-resolution imaging and single-molecule tracking. These results suggest that SGs have a highly organized structure that enables dynamic regulation of the mRNAs at the nanoscale, which is responsible for the ordered formation and the wide variety of functions of SGs.
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The atrophic myocardium resulting from mechanical unloading and nutritional deprivation is considered crucial as maladaptive remodeling directly associated with heart failure, as well as interstitial fibrosis. Conversely, myocardial hypertrophy resulting from hemodynamic loading is perceived as compensatory stress adaptation. We previously reported the abundant presence of highly redox-active polysulfide molecules, termed supersulfide, with two or more sulfur atoms catenated in normal hearts, and the supersulfide catabolism in pathologic hearts after myocardial infarction correlated with worsened prognosis of heart failure. However, the impact of supersulfide on myocardial remodeling remains unclear. Here, we investigated the involvement of supersulfide metabolism in cardiomyocyte remodeling, using a model of adenosine 5'-triphosphate (ATP) receptor-stimulated atrophy and endothelin-1 receptor-stimulated hypertrophy in neonatal rat cardiomyocytes. Results revealed contrasting changes in intracellular supersulfide and its catabolite, hydrogen sulfide (H2S), between cardiomyocyte atrophy and hypertrophy. Stimulation of cardiomyocytes with ATP decreased supersulfide activity, while H2S accumulation itself did not affect cardiomyocyte atrophy. This supersulfide catabolism was also involved in myofibroblast formation of neonatal rat cardiac fibroblasts. Thus, unraveling supersulfide metabolism during myocardial remodeling may lead to the development of novel therapeutic strategies to improve heart failure.
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Sulfeto de Hidrogênio , Miócitos Cardíacos , Sulfetos , Remodelação Ventricular , Animais , Miócitos Cardíacos/metabolismo , Sulfetos/metabolismo , Sulfetos/farmacologia , Sulfeto de Hidrogênio/metabolismo , Células Cultivadas , Trifosfato de Adenosina/metabolismo , Ratos , Atrofia , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Animais Recém-Nascidos , Ratos Sprague-DawleyRESUMO
Senescent cells promote cancer development and progression through chronic inflammation caused by a senescence-associated secretory phenotype (SASP). Although various senotherapeutic strategies targeting senescent cells have been developed for the prevention and treatment of cancers, technology for the in vivo detection and evaluation of senescent cell accumulation has not yet been established. Here, we identified activatable fluorescent probes targeting dipeptidylpeptidase-4 (DPP4) as an effective probe for detecting senescent cells through an enzymatic activity-based screening of fluorescent probes. We also determined that these probes were highly, selectively, and rapidly activated in senescent cells during live cell imaging. Furthermore, we successfully visualized senescent cells in the organs of mice using DPP4-targeted probes. These results are expected to lead to the development of a diagnostic technology for noninvasively detecting senescent cells in vivo and could play a role in the application of DPP4 prodrugs for senotherapy.
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Senescência Celular , Dipeptidil Peptidase 4 , Corantes Fluorescentes , Dipeptidil Peptidase 4/metabolismo , Dipeptidil Peptidase 4/análise , Animais , Camundongos , HumanosRESUMO
9-cyanopyronin is a promising scaffold that exploits resonance Raman enhancement to enable sensitive, highly multiplexed biological imaging. Here, we developed cyano-Hydrol Green (CN-HG) derivatives as resonance Raman scaffolds to expand the color palette of 9-cyanopyronins. CN-HG derivatives exhibit sufficiently long wavelength absorption to produce strong resonance Raman enhancement for near-infrared (NIR) excitation, and their nitrile peaks are shifted to a lower frequency than those of 9-cyanopyronins. The fluorescence of CN-HG derivatives is strongly quenched due to the lack of the 10th atom, unlike pyronin derivatives, and this enabled us to detect spontaneous Raman spectra with high signal-to-noise ratios. CN-HG derivatives are powerful candidates for high performance vibrational imaging.
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Análise Espectral Raman , Estrutura Molecular , Vibração , Nitrilas/química , Nitrilas/síntese químicaRESUMO
Fluorescence imaging is one of the most promising approaches to achieve intraoperative assessment of the tumor/normal tissue margins during cancer surgery. This is critical to improve the patients' prognosis, and therefore various molecular fluorescence imaging probes have been developed for the identification of cancer lesions during surgery. Among them, "activatable" fluorescence probes that react with cancer-specific biomarker enzymes to generate fluorescence signals have great potential for high-contrast cancer imaging due to their low background fluorescence and high signal amplification by enzymatic turnover. Over the past two decades, activatable fluorescence probes employing various fluorescence control mechanisms have been developed worldwide for this purpose. Furthermore, new biomarker enzymatic activities for specific types of cancers have been identified, enabling visualization of various types of cancers with high sensitivity and specificity. This Review focuses on recent advances in the design, function and characteristics of activatable fluorescence probes that target cancer-specific enzymatic activities for cancer imaging and also discusses future prospects in the field of activity-based diagnostics for cancer.
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Neoplasias , Humanos , Fluorescência , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Corantes Fluorescentes , Sondas Moleculares , Imagem Óptica , BiomarcadoresRESUMO
Surgical resection is considered for most brain tumors to obtain tissue diagnosis and to eradicate or debulk the tumor. Glioma, the most common primary malignant brain tumor, generally has a poor prognosis despite the multidisciplinary treatments with radical resection and chemoradiotherapy. Surgical resection of glioma is often complicated by the obscure border between the tumor and the adjacent brain tissues and by the tumor's infiltration into the eloquent brain. 5-aminolevulinic acid is frequently used for tumor visualization, as it exhibits high fluorescence in high-grade glioma. Here, we provide an overview of the fluorescent probes currently used for brain tumors, as well as those under development for other cancers, including HMRG-based probes, 2MeSiR-based probes, and other aminopeptidase probes. We describe our recently developed HMRG-based probes in brain tumors, such as PR-HMRG, combined with the existing diagnosis approach. These probes are remarkably effective for cancer cell recognition. Thus, they can be potentially integrated into surgical treatment for intraoperative detection of cancers.
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Various control strategies are available for building fluorogenic probes to visualize biological events in terms of a fluorescence change. Here, we performed the time-dependent density functional theory (TD-DFT) computational analysis of the twisted intramolecular charge transfer (TICT) process in rhodamine dyes. On the basis of the results, we designed and synthesized a series of rhodamine dyes and established a fluorescence quenching strategy that we call steric repulsion-induced TICT (sr-TICT), in which the fluorescence quenching process is greatly accelerated by simple intramolecular twisting. As proof of concept of this design strategy, we used it to develop a fluorogenic probe, 2-Me PeER (pentyloxyethylrhodamine), for the N-dealkylation activity of CYP3A4. We applied 2-Me PeER for CYP3A4 activity-based fluorescence-activated cell sorting (FACS), providing access to homogeneous, highly functional human-induced pluripotent stem cell (hiPSC)-derived hepatocytes and intestinal epithelial cells. Our results suggest that sr-TICT represents a general fluorescence control method for fluorogenic probes.
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Corantes , Citocromo P-450 CYP3A , Humanos , Fluorescência , Mercaptoetanol , RodaminasRESUMO
Single-molecule enzyme activity-based enzyme profiling (SEAP) is a methodology to globally analyze protein functions in living samples at the single-molecule level. It has been previously applied to detect functional alterations in phosphatases and glycosidases. Here, we expand the potential for activity-based biomarker discovery by developing a semi-automated synthesis platform for fluorogenic probes that can detect various peptidases and protease activities at the single-molecule level. The peptidase/protease probes were prepared on the basis of a 7-amino-4-methylcoumarin fluorophore. The introduction of a phosphonic acid to the core scaffold made the probe suitable for use in a microdevice-based assay, while phosphonic acid served as the handle for the affinity separation of the probe using Phos-tag. Using this semi-automated scheme, 48 fluorogenic probes for the single-molecule peptidase/protease activity analysis were prepared. Activity-based screening using blood samples revealed altered single-molecule activity profiles of CD13 and DPP4 in blood samples of patients with early-stage pancreatic tumors. The study shows the power of single-molecule enzyme activity screening to discover biomarkers on the basis of the functional alterations of proteins.
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Neoplasias Pancreáticas , Peptídeo Hidrolases , Ácidos Fosforosos , Humanos , Peptídeo Hidrolases/metabolismo , Proteínas , Biomarcadores , Hormônios PancreáticosRESUMO
Small-molecule based activatable fluorescence probes for detecting specific enzyme activity with high sensitivity can visualize the expression site of marker genes and cancers where the enzyme is highly expressed. However, the enzyme-catalyzed fluorescent hydrolysis product easily leaks out and diffuses from the reaction site, making it difficult to perform long-term tracking and immunohistochemical analysis which needs washing/fixation procedure. Our group have focused on quinone methide chemistry and developed series of activatable fluorescence probes with excellent intracellular retention that are converted to quinone-methide or aza-quinone-methide intermediates upon reaction with enzymes, which are then react with intracellular nucleophiles such as proteins and glutathione to be retained in cells and to exhibit significant increase in fluorescence. Based on this molecular design, we have developed fluorescence probes targeting ß-galactosidase and γ-glutamyltranspeptidase with different colors. We also developed photo-functional probes such as activatable photosensitizers and caged fluorophores. These probes can visualize or kill target enzyme-expressing cells with high selectivity by suppressing the leakage of hydrolysis products from target cells, and fluorescence imaging in combination with immunostaining was possible due to the high tolerance of the obtained fluorescence signal even after washing and fixation.
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Corantes Fluorescentes , Neoplasias , Humanos , Corantes Fluorescentes/química , Neoplasias/metabolismo , Imagem Óptica/métodos , QuinonasRESUMO
OBJECTIVES: Although the risk of complications due to postoperative pancreatic fistula (POPF) have been evaluated based on the amylase level in drained ascitic fluid, this method has much room for improvement regarding diagnostic accuracy and facility of the measurement. This study aimed to investigate the clinical value of measuring pancreatic chymotrypsin activity for rapid and accurate prediction of POPF after pancreaticoduodenectomy. METHODS: In 52 consecutive patients undergoing pancreaticoduodenectomy, the chymotrypsin activity in pancreatic juice was measured by calculating the increase in fluorescence intensity during the first 5 min after activation with an enzyme-activatable fluorophore. The predictive value for clinically relevant POPF (CR-POPF) was compared between this technique and the conventional method based on the amylase level. RESULTS: According to receiver operating characteristic analyses, pancreatic chymotrypsin activity on postoperative day (POD) 3 measured with a multiplate reader had the highest predictive value for CR-POPF (area under the curve [AUC], 0.752; P < 0.001), yielding 77.8 % sensitivity and 68.8 % specificity. The AUC and sensitivity/specificity of the amylase level in ascitic fluid on POD 3 were 0.695 (P = 0.053) and 77.8 %/41.2 %, respectively. Multivariable analysis identified high pancreatic chymotrypsin activity on POD 3 as an independent risk factor for CR-POPF. Measurement of pancreatic chymotrypsin activity with a prototype portable fluorescence photometer could significantly predict CR-POPF (AUC, 0.731; P = 0.010). CONCLUSION: Measurement of pancreatic chymotrypsin activity enabled accurate and rapid prediction of CR-POPF after pancreaticoduodenectomy. This can help surgeons to implement appropriate drain management at the patient's bedside without delay.
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Quimotripsina , Fístula Pancreática , Humanos , Fístula Pancreática/diagnóstico , Fístula Pancreática/etiologia , Fístula Pancreática/cirurgia , Pâncreas/cirurgia , Pancreaticoduodenectomia/efeitos adversos , Fatores de Risco , Complicações Pós-Operatórias/etiologia , Drenagem/métodos , Amilases , Estudos RetrospectivosRESUMO
Single-molecule enzyme activity assay is a platform that enables the analysis of enzyme activities at single proteoform level. The limitation of the targetable enzymes is the major drawback of the assay, but the general assay platform is reported to study single-molecule enzyme activities of esterases based on the coupled assay using thioesters as substrate analogues. The coupled assay is realized by developing highly water-soluble thiol-reacting probes based on phosphonate-substituted boron dipyrromethene (BODIPY). The system enables the detection of cholinesterase activities in blood samples at single-molecule level, and it is shown that the dissecting alterations of single-molecule esterase activities can serve as an informative platform for activity-based diagnosis.
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Esterases , Esterases/análise , Esterases/químicaRESUMO
Carboxypeptidases (CPs) are a family of hydrolases that cleave one or more amino acids from the C-terminal of peptides or proteins and play indispensable roles in various physiological and pathological processes. However, only a few highly activatable fluorescence probes for CPs have been reported, and there is a need for a flexibly tunable molecular design platform to afford a range of fluorescence probes for CPs for biological and medical research. Here, we focused on the unique activation mechanism of ProTide-based prodrugs and established a modular design platform for CP-targeting florescence probes based on ProTide chemistry. In this design, probe properties such as fluorescence emission wavelength, reactivity/stability, and target CP can be readily tuned and optimized by changing the four probe modules: the fluorophore, the substituent on the phosphorus atom, the linker amino acid at the P1 position, and the substrate amino acid at the P1' position. In particular, switching the linker amino acid at position P1 enabled us to precisely optimize the reactivity for target CPs. As a proof-of-concept, we constructed probes for carboxypeptidase M (CPM) and prostate-specific membrane antigen (also known as glutamate carboxypeptidase II). The developed probes were applicable for the imaging of CP activities in live cells and in clinical specimens from patients. This design strategy should be useful in studying CP-related biological and pathological phenomena.
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Carboxipeptidases , Ariloxifosforamidatos , Masculino , Humanos , Fluorescência , Carboxipeptidases/metabolismo , Hidrolases , Aminoácidos , Corantes Fluorescentes/químicaRESUMO
Introduction: Complete resection is the only possible treatment for cholangiocarcinoma in the extrahepatic biliary tree (eCCA), although current imaging modalities are limited in their ability to accurately diagnose longitudinal spread. We aimed to develop fluorescence imaging techniques for real-time identification of eCCA using an enzyme-activatable probe, which emits fluorescence immediately after activation by a cancer-specific enzyme. Methods: Using lysates and small tissue fragments collected from surgically resected specimens, we selected the most specific probe for eCCA from among 800 enzyme-activatable probes. The selected probe was directly sprayed onto resected specimens and fluorescence images were acquired; these images were evaluated for diagnostic accuracy. We also comprehensively searched for enzymes that could activate the probe, then compared their expression levels in cancer and non-cancer tissues. Results: Analyses of 19 samples (four cancer lysates, seven non-cancer lysates, and eight bile samples) and 54 tissue fragments (13 cancer tissues and 41 non-cancer tissues) revealed that PM-2MeSiR was the most specific fluorophore for eCCA. Fluorescence images of 7 patients were obtained; these images enabled rapid identification of cancerous regions, which closely matched histopathology findings in 4 patients. Puromycin-sensitive aminopeptidase was identified as the enzyme that might activate the probe, and its expression was upregulated in eCCA. Conclusion: Fluorescence imaging with PM-2MeSiR, which may be activated by puromycin-sensitive aminopeptidase, yielded generally high accuracy. This technique may be useful for real-time identification of the spread of eCCA during surgery and endoscopic examinations.
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The purpose of this study is to clarify the metabolic dependence of ovarian clear cell carcinoma (CCC) by comparing normal tissues and to examine the applicability of fluorescence imaging probe to exploit these metabolic differences. Enhanced glutathione synthesis was supported by the increased uptake of related metabolites and elevated expression levels of genes. Accumulation of intracellular iron and lipid peroxide, induction of cell death by inhibition of the glutathione synthesis pathway indicated that ferroptosis was induced. The activation of γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), a fluorescent imaging probe that recognizes γ-glutamyl transferase, which is essential for the synthesis of glutathione, was investigated in fresh-frozen surgical specimens. gGlu-HMRG detected extremely strong fluorescent signals in the tumor lesions of CCC patients, compared to normal ovaries or endometrium. These results revealed that CCC occurs in the stressful and unique environment of free radical-rich endometrioma, and that glutathione metabolism is enhanced as an adaptation to oxidative stress. Furthermore, a modality that exploits these metabolic differences would be useful for distinguishing between CCC and normal tissues.
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Carcinoma , Ovário , Feminino , Humanos , Ovário/metabolismo , Corantes Fluorescentes/metabolismo , Imagem Óptica/métodos , GlutationaRESUMO
D,L-Propargylglycine (PAG) has been widely used as a selective inhibitor to investigate the biological functions of cystathionine γ-lyase (CSE), which catalyzes the formation of reactive sulfur species (RSS). However, PAG also inhibits other PLP (pyridoxal-5'-phosphate)-dependent enzymes such as methionine γ-lyase (MGL) and L-alanine transaminase (ALT), so highly selective CSE inhibitors are still required. Here, we performed high-throughput screening (HTS) of a large chemical library and identified oxamic hydrazide 1 as a potent inhibitor of CSE (IC50 = 13 ± 1 µM (mean ± S.E.)) with high selectivity over other PLP-dependent enzymes and RSS-generating enzymes. Inhibitor 1 inhibited the enzymatic activity of human CSE in living cells, indicating that it is sufficiently membrane-permeable. X-Ray crystal structure analysis of the complex of rat CSE (rCSE) with 1 revealed that 1 forms a Schiff base linkage with the cofactor PLP in the active site of rCSE. PLP in the active site may be a promising target for development of selective inhibitors of PLP-dependent enzymes, including RSS-generating enzymes such as cystathionine ß-synthase (CBS) and cysteinyl-tRNA synthetase 2 (CARS2), which have unique substrate binding pocket structures.
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Cistationina gama-Liase , Bases de Schiff , Animais , Humanos , Ratos , Domínio Catalítico , Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/metabolismo , Fosfatos , Fosfato de Piridoxal/metabolismoRESUMO
The proteasome plays a central role in intracellular protein degradation. Age-dependent decline in proteasome activity is associated with cellular senescence and organismal aging; however, the mechanism by which the proteasome plays a role in senescent cells remains elusive. Here, we show that nuclear foci that contain the proteasome and exhibit liquid-like properties are formed in senescent cells. The formation of senescence-associated nuclear proteasome foci (SANPs) is dependent on ubiquitination and RAD23B, similar to previously known nuclear proteasome foci, but also requires proteasome activity. RAD23B knockdown suppresses SANP formation and increases mitochondrial activity, leading to reactive oxygen species production without affecting other senescence traits such as cell-cycle arrest and cell morphology. These findings suggest that SANPs are an important feature of senescent cells and uncover a mechanism by which the proteasome plays a role in senescent cells.
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Núcleo Celular , Complexo de Endopeptidases do Proteassoma , Complexo de Endopeptidases do Proteassoma/metabolismo , Núcleo Celular/metabolismo , Ubiquitinação , Senescência CelularRESUMO
Irritable bowel syndrome (IBS) has no clinically accepted biomarkers even though it affects a large number of individuals worldwide. To address this lack of understanding, we evaluated peptidase activity in fecal samples from 35 patients with diarrheal IBS without symptom exacerbation (IBS-n) and 35 healthy subjects using a library of 384 fluorescent enzymatic substrate probes. IBS-n patients had high trypsin-like peptidase activity for cleavage of C-terminal lysine and arginine residues and low elastase-like activity for cleavage of C-terminal serine and glycine residues. These fluorescent probe library data, together with diagnostic machine-learning techniques, were able to accurately predict IBS-n. This approach can be used to diagnose diseases where no clinically accepted biomarkers exist, in which fecal enzyme activity is altered and also suggests that the development of new therapies targeting enzyme activities is possible.
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Super-resolution vibrational microscopy is promising to increase the degree of multiplexing of nanometer-scale biological imaging because of the narrower spectral linewidth of molecular vibration compared to fluorescence. However, current techniques of super-resolution vibrational microscopy suffer from various limitations including the need for cell fixation, high power loading, or complicated detection schemes. Here, we present reversible saturable optical Raman transitions (RESORT) microscopy, which overcomes these limitations by using photoswitchable stimulated Raman scattering (SRS). We first describe a bright photoswitchable Raman probe (DAE620) and validate its signal activation and depletion characteristics when exposed to low-power (microwatt level) continuous-wave laser light. By harnessing the SRS signal depletion of DAE620 through a donut-shaped beam, we demonstrate super-resolution vibrational imaging of mammalian cells with excellent chemical specificity and spatial resolution beyond the optical diffraction limit. Our results indicate RESORT microscopy to be an effective tool with high potential for multiplexed super-resolution imaging of live cells.
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Microscopia , Vibração , Animais , Microscopia/métodos , Análise Espectral Raman/métodos , MamíferosRESUMO
The M3 metalloproteases, neurolysin and THOP1, are neuropeptidases that are expressed in various tissues and metabolize neuropeptides, such as neurotensin. The biological roles of these enzymes are not well characterized, partially because the chemical tools to analyse their activities are not well developed. Here, we developed a fluorogenic substrate probe for neurolysin and thimet oligopeptidase 1 (THOP1), which enabled the analysis of enzymatic activity changes in tissue and plasma samples. In particular, the probe was useful for studying enzyme activities in a single-molecule enzyme assay platform, which can detect enzyme activity with high sensitivity. We detected the activity of neurolysin in plasma samples and revealed higher enzyme activity in the blood samples of patients with colorectal tumor. The result indicated that single-molecule neurolysin activity is a promising candidate for a blood biomarker for colorectal cancer diagnosis.
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Controlling tumor-specific alterations in metabolic pathways is a useful strategy for treating tumors. The glyoxalase pathway, which metabolizes the toxic electrophile 2-methylglyoxal (MG), is thought to contribute to tumor pathology. We developed a live cell-based high-throughput screening system that monitors the metabolism of MG to generate D-lactate by glyoxalase I and II (GLO1 and GLO2). It utilizes an extracellular coupled assay that uses D-lactate to generate NAD(P)H, which is detected by a selective fluorogenic probe designed to respond exclusively to extracellular NAD(P)H. This metabolic pathway-oriented screening is able to identify compounds that control MG metabolism in live cells, and we have discovered compounds that can directly or indirectly inhibit glyoxalase activities in small cell lung carcinoma cells.