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1.
J Phys Chem B ; 127(2): 446-455, 2023 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-36607139

RESUMEN

Proteolysis targeting chimera (PROTAC) is a novel drug modality that facilitates the degradation of a target protein by inducing proximity with an E3 ligase. In this work, we present a new computational framework to model the cooperativity between PROTAC-E3 binding and PROTAC-target binding principally through protein-protein interactions (PPIs) induced by the PROTAC. Due to the scarcity and low resolution of experimental measurements, the physical and chemical drivers of these non-native PPIs remain to be elucidated. We develop a coarse-grained (CG) approach to model interactions in the target-PROTAC-E3 complexes, which enables converged thermodynamic estimations using alchemical free energy calculation methods despite an unconventional scale of perturbations. With minimal parametrization, we successfully capture fundamental principles of cooperativity, including the optimality of intermediate PROTAC linker lengths that originates from configurational entropy. We qualitatively characterize the dependency of cooperativity on PROTAC linker lengths and protein charges and shapes. Minimal inclusion of sequence- and conformation-specific features in our current force field, however, limits quantitative modeling to reproduce experimental measurements, but further development of the CG model may allow for efficient computational screening to optimize PROTAC cooperativity.


Asunto(s)
Proteínas , Ubiquitina-Proteína Ligasas , Proteolisis , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas/metabolismo , Termodinámica
2.
Cell Mol Life Sci ; 80(1): 32, 2023 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-36609589

RESUMEN

Protein quality control (PQC) degrons are short protein segments that target misfolded proteins for proteasomal degradation, and thus protect cells against the accumulation of potentially toxic non-native proteins. Studies have shown that PQC degrons are hydrophobic and rarely contain negatively charged residues, features which are shared with chaperone-binding regions. Here we explore the notion that chaperone-binding regions may function as PQC degrons. When directly tested, we found that a canonical Hsp70-binding motif (the APPY peptide) functioned as a dose-dependent PQC degron both in yeast and in human cells. In yeast, Hsp70, Hsp110, Fes1, and the E3 Ubr1 target the APPY degron. Screening revealed that the sequence space within the chaperone-binding region of APPY that is compatible with degron function is vast. We find that the number of exposed Hsp70-binding sites in the yeast proteome correlates with a reduced protein abundance and half-life. Our results suggest that when protein folding fails, chaperone-binding sites may operate as PQC degrons, and that the sequence properties leading to PQC-linked degradation therefore overlap with those of chaperone binding.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteolisis , Pliegue de Proteína , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo
3.
Molecules ; 28(2)2023 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-36677746

RESUMEN

Posttranslational modifications (PTMs) play an important role in cell signaling and they are often deregulated in disease. This review addresses recent advances in the development of heterobifunctional small molecules that enable targeting or hijacking PTMs. This emerging field is spearheaded by proteolysis-targeting chimeras (PROTACs), that induce ubiquitination of their targets and, thus, tag them for degradation by the proteasome. Within the last decade, several improvements have been made to enhance spatiotemporal control of PROTAC-induced degradation as well as cell permeability. Inspired by the success story of PROTACs, additional concepts based on chimeric small molecules have emerged such as phosphatase-recruiting chimeras (PhoRCs). Herein, an overview of strategies causing (de-)phosphorylation, deubiquitination as well as acetylation is provided, and the opportunities and challenges of heterobifunctional molecules for drug discovery are highlighted. Although significant progress has been achieved, a plethora of PTMs have not yet been covered and PTM-inducing chimeras will be helpful tools for chemical biology and could even find application in pharmacotherapy.


Asunto(s)
Descubrimiento de Drogas , Complejo de la Endopetidasa Proteasomal , Proteolisis , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitinación , Transducción de Señal , Inductores de las Enzimas del Citocromo P-450
4.
ACS Chem Biol ; 18(1): 25-33, 2023 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-36606710

RESUMEN

The proteolysis targeting chimera (PROTAC) strategy results in the down-regulation of unwanted protein(s) for disease treatment. In the PROTAC process, a heterobifunctional degrader forms a ternary complex with a target protein of interest (POI) and an E3 ligase, which results in ubiquitination and proteasomal degradation of the POI. While ternary complex formation is a key attribute of PROTAC degraders, modification of the PROTAC molecule to optimize ternary complex formation and protein degradation can be a labor-intensive and tedious process. In this study, we take advantage of DNA-encoded library (DEL) technology to efficiently synthesize a vast number of possible PROTAC molecules and describe a parallel screening approach that utilizes DNA barcodes as reporters of ternary complex formation and cooperative binding. We use a designed PROTAC DEL against BRD4 and CRBN to describe a dual protein affinity selection method and the direct discovery of novel, potent BRD4 PROTACs that importantly demonstrate clear SAR. Such an approach evaluates all the potential PROTACs simultaneously, avoids the interference of PROTAC solubility and permeability, and uses POI and E3 ligase proteins in an efficient manner.


Asunto(s)
Proteínas Nucleares , Factores de Transcripción , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Proteolisis
5.
Int J Mol Sci ; 24(2)2023 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-36675026

RESUMEN

The intracellular retention of mutant cartilage matrix proteins and pathological endoplasmic reticulum (ER) stress disrupts ossification and has been identified as a shared disease mechanism in a range of skeletal dysplasias including short limbed-dwarfism, multiple epiphyseal dysplasia type 5 (EDM5). Although targeting ER stress is an attractive avenue for treatment and has proven successful in the treatment of a related skeletal dysplasia, to date no drugs have proven successful in reducing ER stress in EDM5 caused by the retention of mutant matrilin-3. Our exciting findings show that by using our established luciferase ER stress screening assay, we can identify a "natural" chemical, curcumin, which is able to reduce pathological ER stress in a cell model of EDM5 by promoting the proteasomal degradation mutant matrilin-3. Therefore, this is an important in vitro study in which we describe, for the first time, the success of a naturally occurring chemical as a potential treatment for this currently incurable rare skeletal disease. As studies show that curcumin can be used as a potential treatment for range of diseases in vitro, current research is focused on developing novel delivery strategies to enhance its bioavailability. This is an important and exciting area of research that will have significant clinical impact on a range of human diseases including the rare skeletal disease, EDM5.


Asunto(s)
Condrocitos , Curcumina , Proteínas Matrilinas , Humanos , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Curcumina/farmacología , Curcumina/metabolismo , Estrés del Retículo Endoplásmico , Proteínas Matrilinas/metabolismo , Proteolisis
6.
J Hematol Oncol ; 16(1): 6, 2023 Jan 24.
Artículo en Inglés | MEDLINE | ID: mdl-36694209

RESUMEN

Despite significant progress in clinical management, drug resistance remains a major obstacle. Recent research based on protein degradation to restrain drug resistance has attracted wide attention, and several therapeutic strategies such as inhibition of proteasome with bortezomib and proteolysis-targeting chimeric have been developed. Compared with intervention at the transcriptional level, targeting the degradation process seems to be a more rapid and direct strategy. Proteasomal proteolysis and lysosomal proteolysis are the most critical quality control systems responsible for the degradation of proteins or organelles. Although proteasomal and lysosomal inhibitors (e.g., bortezomib and chloroquine) have achieved certain improvements in some clinical application scenarios, their routine application in practice is still a long way off, which is due to the lack of precise targeting capabilities and inevitable side effects. In-depth studies on the regulatory mechanism of critical protein degradation regulators, including E3 ubiquitin ligases, deubiquitylating enzymes (DUBs), and chaperones, are expected to provide precise clues for developing targeting strategies and reducing side effects. Here, we discuss the underlying mechanisms of protein degradation in regulating drug efflux, drug metabolism, DNA repair, drug target alteration, downstream bypass signaling, sustaining of stemness, and tumor microenvironment remodeling to delineate the functional roles of protein degradation in drug resistance. We also highlight specific E3 ligases, DUBs, and chaperones, discussing possible strategies modulating protein degradation to target cancer drug resistance. A systematic summary of the molecular basis by which protein degradation regulates tumor drug resistance will help facilitate the development of appropriate clinical strategies.


Asunto(s)
Neoplasias , Ubiquitina-Proteína Ligasas , Humanos , Proteolisis , Bortezomib/uso terapéutico , Ubiquitina-Proteína Ligasas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitinación , Chaperonas Moleculares/uso terapéutico , Resistencia a Medicamentos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo
7.
Subcell Biochem ; 102: 99-112, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36600131

RESUMEN

The proteasome is a multi-subunit proteolytic complex that functions to degrade normal proteins for physiological regulation and to eliminate abnormal proteins for cellular protection. Generally, the proteasome targets substrate proteins that are marked by attachment of multiple ubiquitin molecules. In various types of cells in an organism, damage to proteins occurs both from internal sources such as reactive oxygen species and from external ones such as UV radiation from the sun. The proteasome functions to protect the cells by degrading damaged proteins. With ageing, however, the capacity of the proteasome to degrade damaged proteins is reduced as indicated by evidence gathered by many studies. Studies on ageing in muscle, skin, and brain show that with age catalytic activity of the proteasome is decreased and the expression of proteasome subunits is altered. Age-related accumulation of damaged or misfolded proteins causes further reduction of proteasome activity. Abnormal proteins also accumulate as a result of age-related neurodegenerative diseases. Deficits in proteasome activity might be responsible for accumulation of protein aggregates and thus contribute to the pathology. Results from several studies suggest a link between the proteasome and longevity. This chapter reviews the various ways in which the proteasome is associated with the ageing process and examines evidence gathered from investigations on cultured cells, model organisms, and humans.


Asunto(s)
Envejecimiento , Complejo de la Endopetidasa Proteasomal , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Envejecimiento/metabolismo , Proteínas/metabolismo , Ubiquitina/metabolismo , Proteolisis
8.
Theranostics ; 13(1): 374-390, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36593964

RESUMEN

Rationale: The inflammasome has been widely reported to be involved in various myopathies, but little is known about its role in denervated muscle. Here, we explored the role of NLRP3 inflammasome activation in experimental models of denervation in vitro and in vivo. Methods: Employing muscular NLRP3 specific knock-out (NLRP3 cKO) mice, we evaluated the effects of the NLRP3 inflammasome on muscle atrophy in vivo in muscle-specific NLRP3 conditional knockout (cKO) mice subjected to sciatic nerve transection and in vitro in cells incubated with NLRP3 inflammasome activator (NIA). To evaluate the underlying mechanisms, samples were collected at different time points for RNA-sequencing (RNA-seq), and the interacting molecules were comprehensively analysed. Results : In the experimental model, NLRP3 inflammasome activation after denervation led to pyroptosis and upregulation of MuRF1 and Atrogin-1 expression, facilitating ubiquitin-proteasome system (UPS) activation, which was responsible for muscle proteolysis. Conversely, genetic knockout of NLRP3 in muscle inhibited pyroptosis-associated protein expression and significantly ameliorated muscle atrophy. Furthermore, cotreatment with shRNA-NLRP3 markedly attenuated NIA-induced C2C12 myotube pyroptosis and atrophy. Intriguingly, inhibition of NLRP3 inflammasome activation significantly suppressed apoptosis. Conclusions: These in vivo and in vitro findings demonstrate that during denervation, the NLRP3 inflammasome is activated and stimulates muscle atrophy via pyroptosis, proteolysis and apoptosis, suggesting that it may contribute to the pathogenesis of neuromuscular diseases.


Asunto(s)
Inflamasomas , Piroptosis , Ratones , Animales , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteolisis , Apoptosis , Atrofia Muscular/metabolismo , Desnervación
9.
Food Res Int ; 163: 112218, 2023 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-36596147

RESUMEN

The effects of adding different phospholipid (PL) matrices [milk sphingomyelin (SM) vs soy phosphatidylcholine (PC)] on emulsion stability, microstructure, and in vitro simulated lipid digestion were examined using a Model Infant Formula Emulsion (MIFE). The emulsion stability of MIFE increased significantly with PL addition (0.1 and 0.2 %). Compared to sole MIFE or MIFE + PC, the incorporation of SM resulted in increased emulsion stability (p < 0.05) and a greater amount of free fatty acid release (p < 0.05) during in vitro simulated digestion. This was mainly due to the reduction of intensive droplet aggregation, thus providing a large surface area and improved digestibility. This is further experimentally supported by the evolution of particle size distribution, zeta-potential, and microstructure analysis using confocal laser scanning microscopy. The incorporation of SM in the emulsion formation significantly delayed digestion of ß-lactoglobulin during in vitro digestion. Lipid digestibility in MIFE was altered depending on the type of PL matrix, and SM displayed a superior effect to PC. Thus, the creation of a novel emulsion interface by the appropriate selection of emulsifiers can be used to improve lipid digestion in infants and obtain desirable nutritional consequences.


Asunto(s)
Fórmulas Infantiles , Fosfolípidos , Humanos , Emulsiones/química , Proteolisis , Fórmulas Infantiles/química , Emulsionantes
10.
Int J Mol Sci ; 24(1)2023 Jan 03.
Artículo en Inglés | MEDLINE | ID: mdl-36614276

RESUMEN

Auxin action largely depends on the establishment of auxin concentration gradient within plant organs, where PIN-formed (PIN) auxin transporter-mediated directional auxin movement plays an important role. Accumulating studies have revealed the need of polar plasma membrane (PM) localization of PIN proteins as well as regulation of PIN polarity in response to developmental cues and environmental stimuli, amongst which a typical example is regulation of PIN phosphorylation by AGCVIII protein kinases and type A regulatory subunits of PP2A phosphatases. Recent findings, however, highlight the importance of PIN degradation in reestablishing auxin gradient. Although the underlying mechanism is poorly understood, these findings provide a novel aspect to broaden the current knowledge on regulation of polar auxin transport. In this review, we summarize the current understanding on controlling PIN degradation by endosome-mediated vacuolar targeting, autophagy, ubiquitin modification and the related E3 ubiquitin ligases, cytoskeletons, plant hormones, environmental stimuli, and other regulators, and discuss the possible mechanisms according to recent studies.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteolisis , Transporte Biológico , Ácidos Indolacéticos/metabolismo , Ubiquitinas/metabolismo , Raíces de Plantas/metabolismo
11.
J Med Chem ; 66(2): 1339-1348, 2023 Jan 26.
Artículo en Inglés | MEDLINE | ID: mdl-36608275

RESUMEN

While proteolysis-targeting chimeras (PROTACs) are showing promise for targeting previously undruggable molecules, their application has been limited by difficulties in identifying suitable ligands and undesired on-target toxicity. Aptamers can virtually recognize any protein through their unique and switchable conformations. Here, by exploiting aptamers as targeting warheads, we developed a novel strategy for inducible degradation of undruggable proteins. As a proof of concept, we chose oncogenic nucleolin (NCL) as the target and generated a series of NCL degraders, and demonstrated that dNCL#T1 induced NCL degradation in a ubiquitin-proteasome-dependent manner, thereby inhibiting NCL-mediated breast cancer cell proliferation. To reduce on-target toxicity, we further developed a light-controllable PROTAC, opto-dNCL#T1, by introducing a photolabile complementary oligonucleotide to hybridize with dNCL#T1. UVA irradiation liberated dNCL#T1 from caged opto-dNCL#T1, leading to dNCL#T1 activation and NCL degradation. These results indicate that aptamer-based PROTACs are a viable alternative approach to degrade proteins of interest in a highly tunable manner.


Asunto(s)
Fosfoproteínas , Complejo de la Endopetidasa Proteasomal , Proteolisis , Complejo de la Endopetidasa Proteasomal/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo
12.
Sci Adv ; 9(3): eadd3867, 2023 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-36662861

RESUMEN

Successful severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection requires proteolytic cleavage of the viral spike protein. While the role of the host transmembrane protease serine 2 in SARS-CoV-2 infection is widely recognized, the involvement of other proteases capable of facilitating SARS-CoV-2 entry remains incompletely explored. Here, we show that multiple members from the membrane-type matrix metalloproteinase (MT-MMP) and a disintegrin and metalloproteinase families can mediate SARS-CoV-2 entry. Inhibition of MT-MMPs significantly reduces SARS-CoV-2 replication in vitro and in vivo. Mechanistically, we show that MT-MMPs can cleave SARS-CoV-2 spike and angiotensin-converting enzyme 2 and facilitate spike-mediated fusion. We further demonstrate that Omicron BA.1 has an increased efficiency on MT-MMP usage, while an altered efficiency on transmembrane serine protease usage for virus entry compared with that of ancestral SARS-CoV-2. These results reveal additional protease determinants for SARS-CoV-2 infection and enhance our understanding on the biology of coronavirus entry.


Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Péptido Hidrolasas/metabolismo , Proteolisis , Metaloproteasas/metabolismo , Internalización del Virus
13.
Proc Natl Acad Sci U S A ; 120(4): e2216531120, 2023 Jan 24.
Artículo en Inglés | MEDLINE | ID: mdl-36669100

RESUMEN

Executioner-caspase activation has been considered a point-of-no-return in apoptosis. However, numerous studies report survival from caspase activation after treatment with drugs or radiation. An open question is whether cells can recover from direct caspase activation without pro-survival stress responses induced by drugs. To address this question, we engineered a HeLa cell line to express caspase-3 inducibly and combined it with a quantitative caspase activity reporter. While high caspase activity levels killed all cells and very low levels allowed all cells to live, doses of caspase activity sufficient to kill 15 to 30% of cells nevertheless allowed 70 to 85% to survive. At these doses, neither the rate, nor the peak level, nor the total amount of caspase activity could accurately predict cell death versus survival. Thus, cells can survive direct executioner-caspase activation, and variations in cellular state modify the outcome of potentially lethal caspase activity. Such heterogeneities may underlie incomplete tumor cell killing in response to apoptosis-inducing cancer treatments.


Asunto(s)
Apoptosis , Humanos , Supervivencia Celular/fisiología , Células HeLa , Muerte Celular , Apoptosis/fisiología , Caspasa 3/genética , Caspasa 3/metabolismo , Proteolisis , Caspasa 8/metabolismo
14.
Int J Mol Sci ; 24(2)2023 Jan 13.
Artículo en Inglés | MEDLINE | ID: mdl-36675084

RESUMEN

Dry-cured pork products, such as dry-cured ham, undergo an extensive proteolysis during manufacturing process which determines the organoleptic properties of the final product. As a result of endogenous pork muscle endo- and exopeptidases, many medium- and short-chain peptides are released from muscle proteins. Many of them have been isolated, identified, and characterized, and some peptides have been reported to exert relevant bioactivity with potential benefit for human health. However, little attention has been given to di- and tripeptides, which are far less known, although they have received increasing attention in recent years due to their high potential relevance in terms of bioactivity and role in taste development. This review gathers the current knowledge about di- and tripeptides, regarding their bioactivity and sensory properties and focusing on their generation during long-term processing such as dry-cured pork meats.


Asunto(s)
Productos de la Carne , Carne de Cerdo , Carne Roja , Animales , Porcinos , Humanos , Carne de Cerdo/análisis , Péptidos , Proteolisis , Proteínas Musculares/química , Productos de la Carne/análisis , Carne/análisis
15.
Foodborne Pathog Dis ; 20(1): 32-37, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36622956

RESUMEN

The only staphylococcal enterotoxins produced by Staphylococcus epidermidis include SECepi and SELepi, whereas Staphylococcus aureus produces orthologous SECs and SEL having different sequences. We compared S. epidermidis and S. aureus SECs and SELs in terms of resistance to proteolysis and both, thermal and chemical stability. We show that SECepi and SELepi produced by S. epidermidis have similar resistance to proteolysis if compared with their respective orthologues produced by S. aureus. Studied S. epidermidis and S. aureus SEC variants incubated with pepsin at pH 2.0 were found to be more resistant to proteolysis than SELs. SELs turned out to be more resistant than SECs to proteolysis with trypsin at pH 8.0. SECepi was found to be more resistant to thermal denaturation if compared with its S. aureus orthologues. The S. epidermidis and S. aureus SEC variants were found to have higher thermal stability than SELs. Our data indicate that, due to their high stability, the enterotoxins SECepi and SELepi produced in food by S. epidermidis may pose a food safety risk comparable with that posed by S. aureus enterotoxins.


Asunto(s)
Enterotoxinas , Infecciones Estafilocócicas , Humanos , Enterotoxinas/metabolismo , Staphylococcus aureus , Staphylococcus epidermidis/metabolismo , Proteolisis
16.
BMC Microbiol ; 23(1): 9, 2023 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-36627557

RESUMEN

Cytosine deaminase (CDA) is a prodrug mediating enzyme converting 5-flurocytosine into 5-flurouracil with profound broad-range anticancer activity towards various cell lines. Availability, molecular stability, and catalytic efficiency are the main limiting factors halting the clinical applications of this enzyme on prodrug and gene therapies, thus, screening for CDA with unique biochemical and catalytic properties was the objective. Thermotolerant/ thermophilic fungi could be a distinctive repertoire for enzymes with affordable stability and catalytic efficiency. Among the recovered thermotolerant isolates, Aspergillus niger with optimal growth at 45 °C had the highest CDA productivity. The enzyme was purified, with purification 15.4 folds, molecular mass 48 kDa and 98 kDa, under denaturing and native PAGE, respectively. The purified CDA was covalently conjugated with dextran with the highest immobilization yield of 75%. The free and CDA-dextran conjugates have the same optimum pH 7.4, reaction temperature 37 °C, and pI 4.5, and similar response to the inhibitors and amino acids suicide analogues, ensuring the lack of effect of dextran conjugation on the CDA conformational structure. CDA-Dextran conjugates had more resistance to proteolysis in response to proteinase K and trypsin by 2.9 and 1.5 folds, respectively. CDA-Dextran conjugates displayed a dramatic structural and thermal stability than the free enzyme, authenticating the acquired structural and catalytic stability upon dextran conjugation. The thermal stability of CDA was increased by about 1.5 folds, upon dextran conjugation, as revealed from the half-life time (T1/2). The affinity of CDA-conjugates (Km 0.15 mM) and free CDA (Km 0.22 mM) to deaminate 5-fluorocytosine was increased by 1.5 folds. Upon dextran conjugation, the antiproliferative activity of the CDA towards the different cell lines "MDA-MB, HepG-2, and PC-3" was significantly increased by mediating the prodrug 5-FC. The CDA-dextran conjugates strongly reduce the tumor size and weight of the Ehrlich cells (EAC), dramatically increase the titers of Caspase-independent apoptotic markers PARP-1 and AIF, with no cellular cytotoxic activity, as revealed from the hematological and biochemical parameters.


Asunto(s)
Citosina Desaminasa , Profármacos , Humanos , Aspergillus niger , Citosina Desaminasa/metabolismo , Dextranos/metabolismo , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Péptido Hidrolasas/metabolismo , Profármacos/farmacología , Proteolisis , Línea Celular Tumoral
17.
Dis Markers ; 2023: 5899662, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36644609

RESUMEN

Background: Currently, how to successfully control refractory and metastatic diseases remains a fundamental goal for clinicians to improve therapeutic effects for patients with non-small cell lung cancer (NSCLC). Several studies have discovered that TRIM58, a member of tripartite motif protein family, shows antitumor effect in multiple types of cancer. In this study, we aimed to further clarify the molecular regulatory network of TRIM58 and corresponding targets for NSCLC patients. Methods: TRIM58 expression in clinical tumor tissue samples and cancer cell lines was examined. Functional experiments including cellular invasion, cell metastasis, chemoresistance assay, and ubiquitination evaluation experiments were conducted to investigate the interaction between TRIM58 and ZEB1, which is a prime element of transcription factor network that controls epithelial-to-mesenchymal transition. Results: TRIM58 expression was characteristically decreased in NSCLC tumor tissues and cancer cell lines. Functional experiments demonstrated that TRIM58 suppression enhanced malignant biological behaviors including cellular survivability, migration, and invasion, as well as stem-like cellular phenotype of tumor cells. TRIM58 silencing also significantly enhanced the chemoresistance of NSCLC cells to chemoagents. TRIM58-ZEB1 interaction accelerated degradation of ZEB1 protein, thus further leading to the augment of tumor behaviors. Further detailed molecular experiments revealed that the interaction between TRIM58 and ZEB1 was mediated via ubiquitin-proteasome pathway (UPP). Conclusion: TRIM58 suppressed NSCLC through interacting with ZEB1 and promoting ZEB1 protein degradation via UPP. The present research sheds light on the interaction between TRIM58 and ZEB1, and TRIM58/ZEB1 axis might be the potential therapeutic targets of NSCLC.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Proteínas de Motivos Tripartitos , Homeobox 1 de Unión a la E-Box con Dedos de Zinc , Humanos , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Neoplasias Pulmonares/patología , Proteolisis , Ubiquitinación , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismo , Complejo de la Endopetidasa Proteasomal , Ubiquitina
18.
Int J Biol Sci ; 19(2): 377-392, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36632463

RESUMEN

HER2 is a transmembrane receptor with intrinsic tyrosine kinase activity that is overexpressed in almost 25% of human breast cancers. Here, we report that the neddylation of HER2 is a new post-translational modification that controls its expression and oncogenic activity in human breast cancer. Two critical members in the neddylation pathway, NEDD8 and NEDD8-activating enzyme E1 subunit 1 (NAE1), are detected in human breast specimens. Overexpressed NEDD8 and NAE1 are positively correlated with HER2 expression in human breast cancer. Subsequent structure and function experiments show that HER2 directly interacts with NEDD8 and NAE1, whereas HER2 protein expression is decreased by neddylation depletion. Mechanistically, neddylation inhibition promotes the degradation of HER2 protein by improving its ubiquitination. HER2 overexpression abrogates neddylation depletion-triggered cell growth suppression. The inhibition of neddylation synergized with trastuzumab significantly suppresses growth of HER2 positive breast cancer. Collectively, this study demonstrates a previously undiscovered role of NEDD8-dependent HER2 neddylation promotes tumor growth in breast cancer.


Asunto(s)
Neoplasias de la Mama , Humanos , Femenino , Proteolisis , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Ubiquitinas/genética , Ubiquitinas/metabolismo , Procesamiento Proteico-Postraduccional , Ubiquitinación
19.
Sci Adv ; 9(1): eadd6626, 2023 Jan 06.
Artículo en Inglés | MEDLINE | ID: mdl-36608132

RESUMEN

Despite the rapid utilization of immunotherapy, emerging challenges to the current immune checkpoint blockade need to be resolved. Here, we report that elevation of CD73 levels due to its aberrant turnover is correlated with poor prognosis in immune-cold triple-negative breast cancers (TNBCs). We have identified TRIM21 as an E3 ligase that governs CD73 destruction. Disruption of TRIM21 stabilizes CD73 that in turn enhances CD73-catalyzed production of adenosine, resulting in the suppression of CD8+ T cell function. Replacement of lysine 133, 208, 262, and 321 residues by arginine on CD73 attenuated CD73 ubiquitylation and degradation. Diminishing of CD73 ubiquitylation remarkably promotes tumor growth and impedes antitumor immunity. In addition, a TRIM21high/CD73low signature in a subgroup of human breast malignancies was associated with a favorable immune profile. Collectively, our findings uncover a mechanism that governs CD73 proteolysis and point to a new therapeutic strategy by modulating CD73 ubiquitylation.


Asunto(s)
Inmunoterapia , Neoplasias de la Mama Triple Negativas , Humanos , Inmunoterapia/métodos , Linfocitos T CD8-positivos , Proteolisis , Ubiquitina-Proteína Ligasas
20.
Eur J Med Chem ; 247: 115072, 2023 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-36603510

RESUMEN

Targeting Glutathione peroxidase 4 (GPX4) has become a promising strategy for drug-resistant cancer therapy via ferroptosis induction. It was found that the GPX4 inhibitors such as RSL3 have GPX4 degradation ability via not only autophagy-lysosome pathway but also ubiquitin-proteasome system (UPS). Proteolysis targeting chimeras (PROTACs) using small molecule with both inhibition and degradation ability as the ligand of protein of interest (POI) have not been reported. To obtain better compounds with effective disturbance of GPX4 activity, and compare the difference between GPX4 inhibitors with degradation ability and their related PROTACs, we designed and synthesized a series of GPX4 degraders using PROTAC technology in terms of its excellent characteristics such as high efficiency and selectivity and the capacity of overcoming resistance. Hence, 8e was discovered as a potent and highly efficacious GPX4 degrader based upon the inhibitor RSL3. It was 2-3 times more potent than RSL3 in all the in vitro anti-tumor assays, indicating the importance of the PROTAC ternary complex of GPX4, 8e and E3 ligase ligand. 8e revealed better potency in resistant tumor cells than in wide type cells. Furthermore, we discovered for the first time that degrader 8e exhibit GPX4 degradation activity via ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway with UPS plays the major role in the process. Our data also suggested that 8e and RSL3 could potently induce ferroptosis of HT1080 cells via GPX4 inhibition and degradation. In summary, our data revealed that the GPX4 degrader 8e achieves better degradation and anti-tumor effects compared to its related GPX4 inhibitor RSL3. Thus, an efficient strategy to induce GPX4 degradation and subsequent ferroptosis was established in this study for malignant cancer treatment in the future.


Asunto(s)
Ferroptosis , Neoplasias , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Ligandos , Ubiquitinas/metabolismo , Proteolisis
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