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1.
Nat Commun ; 11(1): 3978, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770044

RESUMO

Methionine restriction, a dietary regimen that protects against metabolic diseases and aging, represses cancer growth and improves cancer therapy. However, the response of different cancer cells to this nutritional manipulation is highly variable, and the molecular determinants of this heterogeneity remain poorly understood. Here we report that hepatocyte nuclear factor 4α (HNF4α) dictates the sensitivity of liver cancer to methionine restriction. We show that hepatic sulfur amino acid (SAA) metabolism is under transcriptional control of HNF4α. Knocking down HNF4α or SAA enzymes in HNF4α-positive epithelial liver cancer lines impairs SAA metabolism, increases resistance to methionine restriction or sorafenib, promotes epithelial-mesenchymal transition, and induces cell migration. Conversely, genetic or metabolic restoration of the transsulfuration pathway in SAA metabolism significantly alleviates the outcomes induced by HNF4α deficiency in liver cancer cells. Our study identifies HNF4α as a regulator of hepatic SAA metabolism that regulates the sensitivity of liver cancer to methionine restriction.


Assuntos
Fator 4 Nuclear de Hepatócito/metabolismo , Neoplasias Hepáticas/metabolismo , Metionina/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Cisteína/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator 4 Nuclear de Hepatócito/genética , Fígado/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Mesoderma/efeitos dos fármacos , Mesoderma/patologia , Redes e Vias Metabólicas/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Camundongos , Sorafenibe/farmacologia , Transcrição Genética/efeitos dos fármacos
2.
Phys Rev Lett ; 125(5): 058001, 2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32794838

RESUMO

Glassy, nonexponential relaxations in globular proteins are typically attributed to conformational behaviors that are missing from intrinsically disordered proteins. Yet, we show that single molecules of a disordered-protein construct display two signatures of glassy dynamics, logarithmic relaxations and a Kovacs memory effect, in response to changes in applied tension. We attribute this to the presence of multiple independent local structures in the chain, which we corroborate with a model that correctly predicts the force dependence of the relaxation. The mechanism established here likely applies to other disordered proteins.


Assuntos
Modelos Químicos , Proteínas de Neurofilamentos/química , Cisteína/química , Cinética , Dobramento de Proteína , Termodinâmica
3.
PLoS One ; 15(7): e0232072, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645038

RESUMO

The vasculature within a tumor is highly disordered both structurally and functionally. Endothelial cells that comprise the vasculature are poorly connected causing vessel leakage and exposing the endothelium to a hypoxic microenvironment. Therefore, most anti-angiogenic therapies are generally inefficient and result in acquired resistance to increased hypoxia due to elimination of the vasculature. Recent studies have explored the efficacy of targeting metabolic pathways in tumor cells in combination with anti-angiogenic therapy. However, the metabolic alterations of endothelial cells in response to hypoxia have been relatively unexplored. Here, we measured polar metabolite levels in microvascular endothelial cells exposed to short- and long-term hypoxia with the goal of identifying metabolic vulnerabilities that can be targeted to normalize tumor vasculature and improve drug delivery. We found that many amino acid-related metabolites were altered by hypoxia exposure, especially within alanine-aspartate-glutamate, serine-threonine, and cysteine-methionine metabolism. Additionally, there were significant changes in de novo pyrimidine synthesis as well as glutathione and taurine metabolism. These results provide key insights into the metabolic alterations that occur in endothelial cells in response to hypoxia, which serve as a foundation for future studies to develop therapies that lead to vessel normalization and more efficient drug delivery.


Assuntos
Hipóxia Celular , Células Endoteliais/metabolismo , Redes e Vias Metabólicas , Aminoácidos/metabolismo , Ácido Aspártico/metabolismo , Linhagem Celular , Cisteína/metabolismo , Células HEK293 , Humanos , Microvasos/metabolismo , Nucleotídeos/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(27): 16043-16054, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571919

RESUMO

In the indeterminate nodules of a model legume Medicago truncatula, ∼700 nodule-specific cysteine-rich (NCR) peptides with conserved cysteine signature are expressed. NCR peptides are highly diverse in sequence, and some of these cationic peptides exhibit antimicrobial activity in vitro and in vivo. However, there is a lack of knowledge regarding their structural architecture, antifungal activity, and modes of action against plant fungal pathogens. Here, the three-dimensional NMR structure of the 36-amino acid NCR044 peptide was solved. This unique structure was largely disordered and highly dynamic with one four-residue α-helix and one three-residue antiparallel ß-sheet stabilized by two disulfide bonds. NCR044 peptide also exhibited potent fungicidal activity against multiple plant fungal pathogens, including Botrytis cinerea and three Fusarium spp. It inhibited germination in quiescent spores of B. cinerea In germlings, it breached the fungal plasma membrane and induced reactive oxygen species. It bound to multiple bioactive phosphoinositides in vitro. Time-lapse confocal and superresolution microscopy revealed strong fungal cell wall binding, penetration of the cell membrane at discrete foci, followed by gradual loss of turgor, subsequent accumulation in the cytoplasm, and elevated levels in nucleoli of germlings. Spray-applied NCR044 significantly reduced gray mold disease symptoms caused by the fungal pathogen B. cinerea in tomato and tobacco plants, and postharvest products. Our work illustrates the antifungal activity of a structurally unique NCR peptide against plant fungal pathogens and paves the way for future development of this class of peptides as a spray-on fungistat/fungicide.


Assuntos
Antifúngicos/farmacologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia , Simbiose , Sequência de Aminoácidos , Botrytis/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Cisteína/química , Fusarium/metabolismo , Lycopersicon esculentum/metabolismo , Lycopersicon esculentum/microbiologia , Espectroscopia de Ressonância Magnética , Medicago truncatula/microbiologia , Pichia/metabolismo , Doenças das Plantas/microbiologia , Tabaco/metabolismo , Tabaco/microbiologia
5.
PLoS One ; 15(6): e0234797, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555735

RESUMO

BACKGROUND: Characteristics of patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and cysteine-sparing NOTCH3 mutations are relatively unknown. This study compared clinical and imaging characteristics between patients with CADASIL and cysteine-sparing NOTCH3 mutations and those with CADASIL and cysteine-involving NOTCH3 mutations. METHODS: We retrospectively reviewed medical records of patients with CADASIL admitted to the Asan Medical Center between September 1999 and September 2017. We compared clinical and brain magnetic resonance imaging (MRI) characteristics based on the presence or absence of cysteine-involving NOTCH3 gene mutations. We compared white matter change frequencies and grades in specific spatial regions between the groups according to age-related white matter change (ARWMC) scores. We evaluated the presence, number, and anatomical distributions of cerebral microbleeds according to the microbleed anatomical rating scale. RESULTS: We reviewed data from 79 patients (55 cysteine-involving, 24 cysteine-sparing NOTCH3 mutations). Clinical symptoms and signs did not differ significantly between the groups. The white matter change frequency and ARWMC scores (adjusted for age and stroke risk factors) in the anterior temporal lobes were lower in cysteine-sparing patients than in cysteine-involving patients. Frequencies and grades of the other brain region's white matter changes and cerebral microbleeds were similar between the groups. CONCLUSIONS: Patients with CADASIL and cysteine-sparing NOTCH3 mutations showed less involvement of the anterior temporal lobes in brain MRI than those with CADASIL and cysteine-involving NOTCH3 mutations, although both groups showed similar clinical characteristics.


Assuntos
CADASIL/patologia , Cisteína/genética , Receptor Notch3/genética , Adulto , Idoso , Encéfalo/diagnóstico por imagem , CADASIL/complicações , CADASIL/genética , Feminino , Humanos , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Estudos Retrospectivos , Fatores de Risco , Fatores Sexuais , Acidente Vascular Cerebral/etiologia , Lobo Temporal/metabolismo , Lobo Temporal/patologia
6.
Toxicon ; 184: 158-166, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32569846

RESUMO

Cl13 is a toxin purified previously from the venom of the Mexican scorpion Centruroides limpidus. This toxin affects the function of voltage gated Na+-channels, human subtypes Nav1.4, Nav1.5 and Nav1.6 in a similar manner as other known ß-toxins from scorpion venoms. Here, we report a correction of the primary structure of Cl13, previously published. The peptide does contain 66 amino acids, but residue 58 is a tryptophan and the last C-terminal amino acid is an amidated lysine, instead of arginine. The main contribution of this communication is the determination of the 3D-structure of Cl13, by solution NMR, showing that Cl13 has the classical cysteine-stabilized α/ß (CSα/ß) folding. It has a triple stranded antiparallel beta sheet commonly present in scorpion sodium channel ß-toxins. In addition, we report and discuss a comparison of Cl13 structure with two other toxins (Cn2 and Css2) from scorpions of the same genus Centruroides, which shows important surface similarities with the structure reported here. Finally, the lack of neutralization of Cl13 toxin by two single-chain antibody fragments (scFvs), named LR and 10FG2, which are capable of neutralizing various toxins from Mexican scorpions, is revised. In particular, 10FG2 is capable of neutralizing toxins Cll1 and Cll2 of the same scorpion C. limpidus. The reasons why LR and 10FG2 are unable of neutralizing Cl13 toxin are discussed.


Assuntos
Venenos de Escorpião/química , Sequência de Aminoácidos , Animais , Cisteína , Espectroscopia de Ressonância Magnética , México , Escorpiões
7.
Ecotoxicol Environ Saf ; 200: 110721, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32464438

RESUMO

Glutaredoxins (Grxs) are small (10-15 kDa) glutathione (GSH) - dependent redox proteins. The role of Grxs are well documented in tolerance to heavy metal stress in prokaryotic and mammalian systems and a few plant genera, but is poorly understood in plants against drought. In the present study, two rice glutaredoxin (Osgrx) genes (LOC_Os02g40500 and LOC_Os01g27140) responsible for tolerance against heavy metal stress have been studied for investigating their role against drought. Each glutaredoxin gene was over-expressed in Arabidopsis thaliana to reveal their role in drought stress. The relative expression of both Osgrx genes was higher in the transgenic lines. Transgenic lines of both Osgrxs showed longer roots, higher seed germination, and survival efficiency during drought stress. The physiological parameters (PN, gs, E, WUE, qP, NPQ and ETR), antioxidant enzymes (GRX, GR, GPX, GST, APX, POD, SOD, CAT, DHAR, and MDHAR), antioxidant molecules (ascorbate and GSH) and stress-responsive amino acids (cysteine and proline) levels were additionally increased in transgenic lines of both Osgrxs to provide drought tolerance. The outcomes from this study strongly determined that each Osgrx gene participated in the moderation of drought and might be utilized in biological engineering strategies to overcome drought conditions in different crops.


Assuntos
Glutarredoxinas/genética , Oryza/enzimologia , Estresse Fisiológico , Antioxidantes/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Ácido Ascórbico/metabolismo , Cisteína/metabolismo , Secas , Genes de Plantas , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Prolina/metabolismo , Estresse Fisiológico/genética
8.
Proc Natl Acad Sci U S A ; 117(20): 10789-10796, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32358195

RESUMO

Oxidation of cysteine thiols by physiological reactive oxygen species (ROS) initiates thermogenesis in brown and beige adipose tissues. Cellular selenocysteines, where sulfur is replaced with selenium, exhibit enhanced reactivity with ROS. Despite their critical roles in physiology, methods for broad and direct detection of proteogenic selenocysteines are limited. Here we developed a mass spectrometric method to interrogate incorporation of selenium into proteins. Unexpectedly, this approach revealed facultative incorporation of selenium as selenocysteine or selenomethionine into proteins that lack canonical encoding for selenocysteine. Selenium was selectively incorporated into regulatory sites on key metabolic proteins, including as selenocysteine-replacing cysteine at position 253 in uncoupling protein 1 (UCP1). This facultative utilization of selenium was initiated by increasing cellular levels of organic, but not inorganic, forms of selenium. Remarkably, dietary selenium supplementation elevated facultative incorporation into UCP1, elevated energy expenditure through thermogenic adipose tissue, and protected against obesity. Together, these findings reveal the existence of facultative protein selenation, which correlates with impacts on thermogenic adipocyte function and presumably other biological processes as well.


Assuntos
Tecido Adiposo/metabolismo , Cisteína/metabolismo , Obesidade/metabolismo , Selênio/metabolismo , Termogênese , Proteína Desacopladora 1/metabolismo , Tecido Adiposo/fisiologia , Animais , Células Cultivadas , Masculino , Espectrometria de Massas/métodos , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo
9.
Nat Commun ; 11(1): 2558, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444769

RESUMO

The control of cysteine reactivity is of paramount importance for the synthesis of proteins using the native chemical ligation (NCL) reaction. We report that this goal can be achieved in a traceless manner during ligation by appending a simple N-selenoethyl group to cysteine. While in synthetic organic chemistry the cleavage of carbon-nitrogen bonds is notoriously difficult, we describe that N-selenoethyl cysteine (SetCys) loses its selenoethyl arm in water under mild conditions upon reduction of its selenosulfide bond. Detailed mechanistic investigations show that the cleavage of the selenoethyl arm proceeds through an anionic mechanism with assistance of the cysteine thiol group. The implementation of the SetCys unit in a process enabling the modular and straightforward assembly of linear or backbone cyclized polypeptides is illustrated by the synthesis of biologically active cyclic hepatocyte growth factor variants.


Assuntos
Cisteína/química , Proteínas/síntese química , Compostos de Sulfidrila/química , Oxirredução , Peptídeos/química , Proteínas/química
10.
Biotechniques ; 69(2): 108-112, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32459144

RESUMO

The outbreak of viral pneumonia caused by the novel coronavirus SARS-CoV-2 that began in December 2019 caused high mortality. It has been suggested that the main protease (Mpro) of SARS-CoV-2 may be an important target to discover pharmaceutical compounds for the therapy of this life-threatening disease. Remdesivir, ritonavir and chloroquine have all been reported to play a role in suppressing SARS-CoV-2. Here, we applied a molecular docking method to study the binding stability of these drugs with SARS-CoV-2 Mpro. It appeared that the ligand-protein binding stability of the alliin and SARS-CoV-2 Mpro complex was better than others. The results suggested that alliin may serve as a good candidate as an inhibitor of SARS-CoV-2 Mpro. Therefore, the present research may provide some meaningful guidance for the prevention and treatment of SARS-CoV-2.


Assuntos
Antivirais/farmacologia , Cisteína/análogos & derivados , Proteínas não Estruturais Virais/antagonistas & inibidores , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Antimaláricos/farmacologia , Betacoronavirus/enzimologia , Cloroquina/farmacologia , Cisteína/farmacologia , Cisteína Endopeptidases , Simulação de Acoplamento Molecular , Ritonavir/farmacologia
11.
Proc Natl Acad Sci U S A ; 117(22): 12101-12108, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414921

RESUMO

Membrane anchoring of farnesylated KRAS is critical for activation of RAF kinases, yet our understanding of how these proteins interact on the membrane is limited to isolated domains. The RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF engage KRAS and the plasma membrane, unleashing the kinase domain from autoinhibition. Due to experimental challenges, structural insight into this tripartite KRAS:RBD-CRD:membrane complex has relied on molecular dynamics simulations. Here, we report NMR studies of the KRAS:CRAF RBD-CRD complex. We found that the nucleotide-dependent KRAS-RBD interaction results in transient electrostatic interactions between KRAS and CRD, and we mapped the membrane interfaces of the CRD, RBD-CRD, and the KRAS:RBD-CRD complex. RBD-CRD exhibits dynamic interactions with the membrane through the canonical CRD lipid-binding site (CRD ß7-8), as well as an alternative interface comprising ß6 and the C terminus of CRD and ß2 of RBD. Upon complex formation with KRAS, two distinct states were observed by NMR: State A was stabilized by membrane association of CRD ß7-8 and KRAS α4-α5 while state B involved the C terminus of CRD, ß3-5 of RBD, and part of KRAS α5. Notably, α4-α5, which has been proposed to mediate KRAS dimerization, is accessible only in state B. A cancer-associated mutation on the state B membrane interface of CRAF RBD (E125K) stabilized state B and enhanced kinase activity and cellular MAPK signaling. These studies revealed a dynamic picture of the assembly of the KRAS-CRAF complex via multivalent and dynamic interactions between KRAS, CRAF RBD-CRD, and the membrane.


Assuntos
Membrana Celular/metabolismo , Cisteína/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sítios de Ligação , Cisteína/química , Humanos , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética
12.
Proc Natl Acad Sci U S A ; 117(20): 10989-10999, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32354997

RESUMO

Staphylococcus aureus infections can lead to diseases that range from localized skin abscess to life-threatening toxic shock syndrome. The SrrAB two-component system (TCS) is a global regulator of S. aureus virulence and critical for survival under environmental conditions such as hypoxic, oxidative, and nitrosative stress found at sites of infection. Despite the critical role of SrrAB in S. aureus pathogenicity, the mechanism by which the SrrAB TCS senses and responds to these environmental signals remains unknown. Bioinformatics analysis showed that the SrrB histidine kinase contains several domains, including an extracellular Cache domain and a cytoplasmic HAMP-PAS-DHp-CA region. Here, we show that the PAS domain regulates both kinase and phosphatase enzyme activity of SrrB and present the structure of the DHp-CA catalytic core. Importantly, this structure shows a unique intramolecular cysteine disulfide bond in the ATP-binding domain that significantly affects autophosphorylation kinetics. In vitro data show that the redox state of the disulfide bond affects S. aureus biofilm formation and toxic shock syndrome toxin-1 production. Moreover, with the use of the rabbit infective endocarditis model, we demonstrate that the disulfide bond is a critical regulatory element of SrrB function during S. aureus infection. Our data support a model whereby the disulfide bond and PAS domain of SrrB sense and respond to the cellular redox environment to regulate S. aureus survival and pathogenesis.


Assuntos
Proteínas de Bactérias/metabolismo , Cisteína/metabolismo , Proteínas Repressoras/metabolismo , Staphylococcus aureus/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Toxinas Bacterianas , Sequência de Bases , Biofilmes , Domínio Catalítico , Modelos Animais de Doenças , Endocardite , Enterotoxinas , Feminino , Regulação Bacteriana da Expressão Gênica , Histidina Quinase/metabolismo , Masculino , Modelos Moleculares , Mutação , Oxirredução , Domínios Proteicos , Coelhos , Proteínas Repressoras/química , Proteínas Repressoras/genética , Sepse , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Superantígenos , Thermotoga maritima , Virulência/genética , Virulência/fisiologia
13.
Cancer Immunol Immunother ; 69(9): 1869-1880, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32372139

RESUMO

Increased proteolytic activity of cysteine cathepsins has long been known to facilitate malignant progression, and it has also been associated with tumor-promoting roles of myeloid-derived suppressor cells (MDSCs). Consequently, cysteine cathepsins have gained much attention as potential targets for cancer therapies. However, cross-talk between tumor cells and MDSCs needs to be taken into account when studying the efficacy of cathepsin inhibitors as anti-cancer agents. Here, we demonstrate the potential of the MDA-MB-231 breast cancer cell line to generate functional MDSCs from CD14+ cells of healthy human donors. During this transition to MDSCs, the overall levels of cysteine cathepsins increased, with the largest responses for cathepsins L and X. We used small-molecule inhibitors of cathepsins L and X (i.e., CLIK-148, Z9, respectively) to investigate their functional impact on tumor cells and immune cells in this co-culture system. Interactions with peripheral blood mononuclear cells reduced MDA-MB-231 cell invasion, while inhibition of cathepsin X activity by Z9 restored invasion. Inhibition of cathepsin L activity using CLIK-148 resulted in significantly increased CD8+ cytotoxicity. Of note, inhibition of cathepsins L and X in separate immune or tumor cells did not promote these functional changes. Together, our findings underlie the importance of tumor cell-immune cell interactions in the evaluation of the anti-cancer potential of cysteine cathepsin inhibitors.


Assuntos
Catepsina L/metabolismo , Cisteína/metabolismo , Células Supressoras Mieloides/metabolismo , Neoplasias/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Humanos , Leucócitos Mononucleares/metabolismo , Invasividade Neoplásica/patologia , Neoplasias/patologia , Células PC-3
14.
Int J Nanomedicine ; 15: 2765-2776, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32425520

RESUMO

Purpose: Over the past decades, quantum dots (QDs) have shown the broad application in diverse fields, especially in intracellular probing and drug delivery, due to their high fluorescence intensity, long fluorescence lifetime, strong light-resistant bleaching ability, and strong light stability. Therefore, we explore a kind of therapeutic potential against cancer with fluorescent imaging. Methods: In the current study, a new type of QDs (QDs@L-Cys-TAEA-5-FUA) capped with L-cysteine (L-Cys) and tris(2-aminoethyl)amine (TAEA) ligands, and conjugated with 5-fluorouracil-1-acetic acid (5-FUA) has been synthesized. Ligands were characterized by electrospray ionization mass spectrometry and H-nuclear magnetic resonance (1H NMR) spectroscopy. The modified QDs were characterized by transmission electron microscopy, ultraviolet and visible spectrophotometry (UV-Vis), and fluorescence microscopy. And the biological activity of modified QDs was explored by using MTT assay with HeLa, SMMC-7721 HepG2, and QSG-7701 cells. The fluorescence imaging of modified QDs was obtained by fluorescence microscope. Results: The modified QDs are of controllable sizes in the range of 4-5 nm and they possess strong optical emission properties. UV-Vis and fluorescence spectra demonstrated that the L-Cys-TAEA-5-FUA was successfully incorporated into QD nanoparticles. The MTT results demonstrated that L-Cys-TAEA-5-FUA modified QDs could efficiently inhibit the proliferation of cancer cells as compared to the normal cells, illustrating their antitumor efficacy. The mechanistic studies revealed that the effective internalization of modified QDs inside cancer cells could inhibit their proliferation, through excessive production of intracellular reactive oxygen species, leading to apoptosis process. Conclusion: The present study suggests that modified QDs can enter cells efficiently and could be employed as therapeutic agents for the treatment of various types of cancers with fluorescent imaging.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Fluoruracila/farmacologia , Pontos Quânticos/química , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Cisteína/química , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Etilenodiaminas/química , Fluoruracila/administração & dosagem , Fluoruracila/química , Células HeLa , Células Hep G2 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Pontos Quânticos/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Espectrofotometria Ultravioleta
15.
Plant Foods Hum Nutr ; 75(3): 376-382, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32399667

RESUMO

The composition of garlic (Allium sativum L.) may vary among cultivars and, moreover, change over time, thereby affecting both biological activity and flavour. Thus, it is important to identify the trends in the content of bioactive compounds in garlic, by reliable analytical methods. This study was focused on the key sulfur-containing compounds, S-alk(en)yl-L-cysteine sulfoxides (alliin, isoalliin, methiin, propiin), which were quantified by a fast liquid chromatography - tandem mass spectrometry (LC-MS/MS) method. Several garlic cultivars were monitored repeatedly within seven months: one month before harvest maturity; at harvest maturity; and after two and six months of storage. The results showed not only a high variability among individual cultivars, but also among samples of the same cultivar grown at different localities. During storage, a significant increase in isoalliin content (up to 54-fold after six months) occurred. Nevertheless, none of the cultivars showed significantly different properties compared to others, suggesting that many other factors affect garlic composition.


Assuntos
Alho , Cromatografia Líquida , Cisteína , Sulfóxidos , Espectrometria de Massas em Tandem
16.
PLoS Biol ; 18(4): e3000656, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32271748

RESUMO

Chemokines and their receptors are orchestrators of cell migration in humans. Because dysregulation of the receptor-chemokine system leads to inflammation and cancer, both chemokines and receptors are highly sought therapeutic targets. Yet one of the barriers for their therapeutic targeting is the limited understanding of the structural principles behind receptor-chemokine recognition and selectivity. The existing structures do not include CXC subfamily complexes and lack information about the receptor distal N-termini, despite the importance of the latter in signaling, regulation, and bias. Here, we report the discovery of the geometry of the complex between full-length CXCR4, a prototypical CXC receptor and driver of cancer metastasis, and its endogenous ligand CXCL12. By comprehensive disulfide cross-linking, we establish the existence and the structure of a novel interface between the CXCR4 distal N-terminus and CXCL12 ß1-strand, while also recapitulating earlier findings from nuclear magnetic resonance, modeling and crystallography of homologous receptors. A cross-linking-informed high-resolution model of the CXCR4-CXCL12 complex pinpoints the interaction determinants and reveals the occupancy of the receptor major subpocket by the CXCL12 proximal N terminus. This newly found positioning of the chemokine proximal N-terminus provides a structural explanation of CXC receptor-chemokine selectivity against other subfamilies. Our findings challenge the traditional two-site understanding of receptor-chemokine recognition, suggest the possibility of new affinity and signaling determinants, and fill a critical void on the structural map of an important class of therapeutic targets. These results will aid the rational design of selective chemokine-receptor targeting small molecules and biologics with novel pharmacology.


Assuntos
Quimiocina CXCL12/química , Quimiocina CXCL12/metabolismo , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Animais , Sítios de Ligação , Western Blotting , Quimiocina CXCL12/genética , Cisteína/química , Cisteína/genética , Dissulfetos/química , Citometria de Fluxo , Células HEK293 , Humanos , Insetos/citologia , Modelos Moleculares , Mutação , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Receptores CXCR4/genética , beta-Arrestinas/metabolismo
17.
Biochim Biophys Acta Proteins Proteom ; 1868(7): 140422, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32234550

RESUMO

As a widespread and reversible post-translational modification of proteins, S-glutathionylation specifically generates the mixed disulfides between cysteine residues and glutathione, which regulates various biological processes including oxidative stress, nitrosative stress and signal transduction. The identification of proteins and specific sites that undergo S-glutathionylation is crucial for understanding the underlying mechanisms and regulatory effects of S-glutathionylation. Experimental identification of S-glutathionylation sites is laborious and time-consuming, whereas computational predictions are more attractive due to their high speed and convenience. Here, we developed a novel computational framework DeepGSH (http://deepgsh.cancerbio.info/) for species-specific S-glutathionylation sites prediction, based on deep learning and particle swarm optimization algorithms. 5-fold cross validation indicated that DeepGSH was able to achieve an AUC of 0.8393 and 0.8458 for Homo sapiens and Mus musculus. According to critical evaluation and comparison, DeepGSH showed excellent robustness and better performance than existing tools in both species, demonstrating DeepGSH was suitable for S-glutathionylation prediction. The prediction results of DeepGSH might provide guidance for experimental validation of S-glutathionylation sites and helpful information to understand the intrinsic mechanisms.


Assuntos
Aprendizado Profundo , Proteína S/química , Proteína S/metabolismo , Algoritmos , Animais , Cisteína/metabolismo , Glutationa/metabolismo , Humanos , Camundongos , Processamento de Proteína Pós-Traducional , Especificidade da Espécie
18.
Biochim Biophys Acta Proteins Proteom ; 1868(7): 140423, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32247787

RESUMO

The human genome encodes for 11 papain-like endolysosomal cysteine peptidases, collectively known as the cysteine cathepsins. Based on their biochemical properties and with the help of experiments in cell culture, the cysteine cathepsins have acquired a reputation as promotors of progression and metastasis of various cancer entities. However, tumors are known to be complex tissues in which non-cancerous cells are also critical for tumorigenesis. Here we discuss the results of the intense investigation of cathepsins in mouse models of human cancers. We focus on models in immunocompetent mice, because only such models allow for analysis of cathepsins in a fully functional tumor microenvironment. An important outcome of those studies was the identification of cancer-promoting cathepsins in tumor-associated macrophages. Another interesting outcome of these animal studies was the identification of a homeostatic tumor-suppressive role for cathepsin L in skin and intestinal cancers. Taken together, these in vivo findings provide a basis for the use of cysteine cathepsins as therapeutic targets, prodrug activators, or as proteases for imaging tumors.


Assuntos
Catepsinas/metabolismo , Cisteína/metabolismo , Neoplasias/metabolismo , Microambiente Tumoral/fisiologia , Aloenxertos , Animais , Neoplasias da Mama/metabolismo , Carcinogênese , Catepsina B/genética , Catepsina B/metabolismo , Catepsina L , Catepsinas/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Intestinais/metabolismo , Camundongos , Neoplasias/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Cutâneas/metabolismo , Microambiente Tumoral/genética
19.
Artigo em Inglês | MEDLINE | ID: mdl-32224438

RESUMO

A fusion peptide mimicking a part of the sequence of HIV-1 envelope glycoprotein with an additional cysteine at its C-terminus (FP8: AVGIGAVFC) was conjugated to a carrier protein through a linker for development of an HIV-1 vaccine. Since this fusion peptide is very hydrophobic with poor solubility and can self-dimerize via a disulfide bond, co-existence of monomeric and dimeric forms presented a major challenge for residual unconjugated FP8 quantification. A reversed-phase liquid chromatography (RPLC) with UV detection was developed to monitor residual FP8 using an experimental correction factor of 0.85 for UV peak area measurement between FP8 dimer and monomer. Therefore, both forms of unconjugated residual FP8 can be measured based on a single FP8 monomer reference curve. Overall, this study demonstrated that the current purification process can remove free residual FP8 to a low level, <20 µg/mL, which showed negligible impact (<10%) for the conjugated FP8 ratio measurement using another method, amino acid analysis.


Assuntos
Peptídeos/análise , Proteínas Recombinantes de Fusão/análise , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Cisteína/química , Dissulfetos/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção , Conformação Proteica , Multimerização Proteica
20.
Proc Natl Acad Sci U S A ; 117(19): 10593-10602, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32332165

RESUMO

A physiological role for long-chain acyl-CoA esters to activate ATP-sensitive K+ (KATP) channels is well established. Circulating palmitate is transported into cells and converted to palmitoyl-CoA, which is a substrate for palmitoylation. We found that palmitoyl-CoA, but not palmitic acid, activated the channel when applied acutely. We have altered the palmitoylation state by preincubating cells with micromolar concentrations of palmitic acid or by inhibiting protein thioesterases. With acyl-biotin exchange assays we found that Kir6.2, but not sulfonylurea receptor (SUR)1 or SUR2, was palmitoylated. These interventions increased the KATP channel mean patch current, increased the open time, and decreased the apparent sensitivity to ATP without affecting surface expression. Similar data were obtained in transfected cells, rat insulin-secreting INS-1 cells, and isolated cardiac myocytes. Kir6.2ΔC36, expressed without SUR, was also positively regulated by palmitoylation. Mutagenesis of Kir6.2 Cys166 prevented these effects. Clinical variants in KCNJ11 that affect Cys166 had a similar gain-of-function phenotype, but was more pronounced. Molecular modeling studies suggested that palmitoyl-C166 and selected large hydrophobic mutations make direct hydrophobic contact with Kir6.2-bound PIP2 Patch-clamp studies confirmed that palmitoylation of Kir6.2 at Cys166 enhanced the PIP2 sensitivity of the channel. Physiological relevance is suggested since palmitoylation blunted the regulation of KATP channels by α1-adrenoreceptor stimulation. The Cys166 residue is conserved in some other Kir family members (Kir6.1 and Kir3, but not Kir2), which are also subject to regulated palmitoylation, suggesting a general mechanism to control the open state of certain Kir channels.


Assuntos
Canais KATP/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Acil Coenzima A/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cisteína/metabolismo , Células HEK293 , Humanos , Canais KATP/genética , Lipoilação/fisiologia , Mutagênese/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp/métodos , Canais de Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Cultura Primária de Células , Ratos , Receptores Sulfonilureia/genética
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