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1.
Eur J Pharm Biopharm ; 201: 114354, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38852755

RESUMO

One of the most appealing approaches for cancer treatment is targeted therapy, which is based on the use of drugs able to target cancer cells without affecting normal ones. This strategy lets to overcome the major limitation of conventional chemotherapy, namely the lack of specificity of anticancer drugs, which often leads to severe side effects, decreasing the therapy effectiveness. Delivery of cell-killing substances to tumor cells is one-way targeted drug therapy can work. Generally, monoclonal antibodies are combined with chemotherapeutic drugs, allowing cellular uptake through the binding to their targets on the surface of cancer cells. Aptamer-drug conjugates represent a promising alternative solution to antibodies to minimize off-target effects, considering the remarkable selective binding capabilities of aptamers. In this study, to enhance the therapeutic efficacy of the antineoplastic agent 5-fluoro-2'-deoxyuridine (FdU) in various cancer cells, we focused on the development of a novel conjugate using the antiproliferative aptamer T30923 (INT) as a drug vehicle. Three derivatives composed of T30923 conjugated with a different number of FdU units were synthesized, and their structural and biological properties were thoroughly characterized, highlighting their potential for targeted and synergistic anticancer responses.


Assuntos
Antineoplásicos , Aptâmeros de Nucleotídeos , Proliferação de Células , Desoxiuridina , Sinergismo Farmacológico , Humanos , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/farmacologia , Aptâmeros de Nucleotídeos/administração & dosagem , Antineoplásicos/farmacologia , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Linhagem Celular Tumoral , Desoxiuridina/análogos & derivados , Desoxiuridina/administração & dosagem , Desoxiuridina/farmacologia , Desoxiuridina/química , Proliferação de Células/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos
2.
Exp Hematol Oncol ; 13(1): 38, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38581035

RESUMO

Rhabdomyosarcoma (RMS), such as other childhood tumors, has witnessed treatment advancements in recent years. However, high-risk patients continue to face poor survival rates, often attributed to the presence of the PAX3/7-FOXO1 fusion proteins, which has been associated with metastasis and treatment resistance. Despite efforts to directly target these chimeric proteins, clinical success remains elusive. In this study, the main aim was to address this challenge by investigating regulators of FOXO1. Specifically, we focused on TRIB3, a potential regulator of the fusion protein in RMS. Our findings revealed a prominent TRIB3 expression in RMS tumors, highlighting its correlation with the presence of fusion protein. By conducting TRIB3 genetic inhibition experiments, we observed an impairment on cell proliferation. Notably, the knockdown of TRIB3 led to a decrease in PAX3-FOXO1 and its target genes at protein level, accompanied by a reduction in the activity of the Akt signaling pathway. Additionally, inducible silencing of TRIB3 significantly delayed tumor growth and improved overall survival in vivo. Based on our analysis, we propose that TRIB3 holds therapeutic potential for treating the most aggressive subtype of RMS. The findings herein reported contribute to our understanding of the underlying molecular mechanisms driving RMS progression and provide novel insights into the potential use of TRIB3 as a therapeutic intervention for high-risk RMS patients.

3.
PNAS Nexus ; 3(1): pgae006, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38269070

RESUMO

A number of intrinsically disordered proteins (IDPs) encoded in stress-tolerant organisms, such as tardigrade, can confer fitness advantage and abiotic stress tolerance when heterologously expressed. Tardigrade-specific disordered proteins including the cytosolic-abundant heat-soluble proteins are proposed to confer stress tolerance through vitrification or gelation, whereas evolutionarily conserved IDPs in tardigrades may contribute to stress tolerance through other biophysical mechanisms. In this study, we characterized the mechanism of action of an evolutionarily conserved, tardigrade IDP, HeLEA1, which belongs to the group-3 late embryogenesis abundant (LEA) protein family. HeLEA1 homologs are found across different kingdoms of life. HeLEA1 is intrinsically disordered in solution but shows a propensity for helical structure across its entire sequence. HeLEA1 interacts with negatively charged membranes via dynamic disorder-to-helical transition, mainly driven by electrostatic interactions. Membrane interaction of HeLEA1 is shown to ameliorate excess surface tension and lipid packing defects. HeLEA1 localizes to the mitochondrial matrix when expressed in yeast and interacts with model membranes mimicking inner mitochondrial membrane. Yeast expressing HeLEA1 shows enhanced tolerance to hyperosmotic stress under nonfermentative growth and increased mitochondrial membrane potential. Evolutionary analysis suggests that although HeLEA1 homologs have diverged their sequences to localize to different subcellular organelles, all homologs maintain a weak hydrophobic moment that is characteristic of weak and reversible membrane interaction. We suggest that such dynamic and weak protein-membrane interaction buffering alterations in lipid packing could be a conserved strategy for regulating membrane properties and represent a general biophysical solution for stress tolerance across the domains of life.

4.
Nanomedicine ; 55: 102722, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38007069

RESUMO

DNA nanostructures have captured great interest as drug delivery vehicles for cancer therapy. Despite rapid progress in the field, some hurdles, such as low cellular uptake, low tissue specificity or ambiguous drug loading, remain unsolved. Herein, well-known antitumor drugs (doxorubicin, auristatin, and floxuridine) were site-specifically incorporated into DNA nanostructures, demonstrating the potential advantages of covalently linking drug molecules via structural staples instead of incorporating the drugs by noncovalent binding interactions. The covalent strategy avoids critical issues such as an unknown number of drug-DNA binding events and premature drug release. Moreover, covalently modified origami offers the possibility of precisely incorporating several synergetic antitumor drugs into the DNA nanostructure at a predefined molar ratio and to control the exact spatial orientation of drugs into DNA origami. Additionally, DNA-based nanoscaffolds have been reported to have a low intracellular uptake. Thus, two cellular uptake enhancing mechanisms were studied: the introduction of folate units covalently linked to DNA origami and the transfection of DNA origami with Lipofectamine. Importantly, both methods increased the internalization of DNA origami into HTB38 and HCC2998 colorectal cancer cells and produced greater cytotoxic activity when the DNA origami incorporated antiproliferative drugs. The results here present a successful and conceptually distinct approach for the development of DNA-based nanostructures as drug delivery vehicles, which can be considered an important step towards the development of highly precise nanomedicines.


Assuntos
Antineoplásicos , Nanoestruturas , Neoplasias , Antineoplásicos/farmacologia , DNA/química , Sistemas de Liberação de Medicamentos , Doxorrubicina/farmacologia , Doxorrubicina/química , Nanoestruturas/química , Conformação de Ácido Nucleico , Nanotecnologia
5.
J Am Chem Soc ; 145(41): 22287-22292, 2023 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-37774000

RESUMO

Protein palmitoylation, with more than 5000 substrates, is the most prevalent form of protein lipidation. Palmitoylated proteins participate in almost all areas of cellular physiology and have been linked to several human diseases. Twenty-three zDHHC enzymes catalyze protein palmitoylation with extensive overlap among the substrates of each zDHHC member. Currently, there is no global strategy to delineate the physiological substrates of individual zDHHC enzymes without perturbing the natural cellular pool. Here, we outline a general approach to accomplish this on the basis of synthetic orthogonal substrates that are only compatible with engineered zDHHC enzymes. We demonstrate the utility of this strategy by validating known substrates and use it to identify novel substrates of two human zDHHC enzymes. Finally, we employ this method to discover and explore conserved palmitoylation in a family of host restriction factors against pathogenic viruses, including SARS-CoV-2.


Assuntos
Aciltransferases , COVID-19 , Humanos , Aciltransferases/metabolismo , Especificidade por Substrato , SARS-CoV-2/metabolismo , Proteínas/metabolismo , Lipoilação
6.
Cancers (Basel) ; 15(6)2023 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-36980521

RESUMO

The identification of novel therapeutic targets for specific cancer molecular subtypes is crucial for the development of precision oncology. In the last few years, CRISPR/Cas9 screens have accelerated the discovery and validation of new targets associated with different tumor types, mutations, and fusions. However, there are still many cancer vulnerabilities associated with specific molecular features that remain to be explored. Here, we used data from CRISPR/Cas9 screens in 954 cancer cell lines to identify gene dependencies associated with 16 common cancer genomic amplifications. We found that high-copy-number genomic amplifications generate multiple collateral dependencies within the amplified region in most cases. Further, to prioritize candidate targets for each chromosomal region amplified, we integrated gene dependency parameters with both druggability data and subcellular location. Finally, analysis of the relationship between gene expression and gene dependency led to the identification of genes, the expression of which may constitute predictive biomarkers of dependency. In conclusion, our study provides a set of druggable targets specific for each amplification, opening the possibility to specifically target amplified tumors on this basis.

7.
Pharmaceutics ; 15(2)2023 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-36839642

RESUMO

Antisense and small interfering RNA (siRNA) oligonucleotides have been recognized as powerful therapeutic compounds for targeting mRNAs and inducing their degradation. However, a major obstacle is that unmodified oligonucleotides are not readily taken up into tissues and are susceptible to degradation by nucleases. For these reasons, the design and preparation of modified DNA/RNA derivatives with better stability and an ability to be produced at large scale with enhanced uptake properties is of vital importance to improve current limitations. In the present study, we review the conjugation of oligonucleotides with lipids and peptides in order to produce oligonucleotide conjugates for therapeutics aiming to develop novel compounds with favorable pharmacokinetics.

8.
Sci Adv ; 9(6): eade9238, 2023 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-36753540

RESUMO

Rhabdomyosarcoma (RMS) is a group of pediatric cancers with features of developing skeletal muscle. The cellular hierarchy and mechanisms leading to developmental arrest remain elusive. Here, we combined single-cell RNA sequencing, mass cytometry, and high-content imaging to resolve intratumoral heterogeneity of patient-derived primary RMS cultures. We show that the aggressive alveolar RMS (aRMS) subtype contains plastic muscle stem-like cells and cycling progenitors that drive tumor growth, and a subpopulation of differentiated cells that lost its proliferative potential and correlates with better outcomes. While chemotherapy eliminates cycling progenitors, it enriches aRMS for muscle stem-like cells. We screened for drugs hijacking aRMS toward clinically favorable subpopulations and identified a combination of RAF and MEK inhibitors that potently induces myogenic differentiation and inhibits tumor growth. Overall, our work provides insights into the developmental states underlying aRMS aggressiveness, chemoresistance, and progression and identifies the RAS pathway as a promising therapeutic target.


Assuntos
Antineoplásicos , Rabdomiossarcoma Alveolar , Rabdomiossarcoma , Criança , Humanos , Rabdomiossarcoma Alveolar/tratamento farmacológico , Rabdomiossarcoma Alveolar/genética , Rabdomiossarcoma Alveolar/patologia , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Músculo Esquelético/metabolismo , Diferenciação Celular , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral
9.
Allergy ; 78(1): 202-213, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-35841381

RESUMO

BACKGROUND: Anaphylaxis is the most acute and life-threatening manifestation of allergic disorders. Currently, there is a need to improve its medical management and increase the understanding of its molecular mechanisms. This study aimed to quantify the extravasation underlying human anaphylactic reactions and propose new theragnostic approaches. METHODS: Molecular determinations were performed in paired serum samples obtained during the acute phase and at baseline from patients presenting with hypersensitivity reactions. These were classified according to their severity as Grades 1, 2 and 3, the two latter being considered anaphylaxis. Tryptase levels were measured by ImmunoCAP, and serum protein concentration was quantified by Bradford assay. Human serum albumin (HSA) and haemoglobin beta subunit (HBB) levels were determined by Western blot and polyacrylamide gel electrophoresis, respectively. RESULTS: A total of 150 patients were included in the study. Of them, 112 had experienced anaphylaxis (83 and 29 with Grade 2 and 3 reactions, respectively). Tryptase diagnostic efficiency substantially improved when considering patients' baseline values (33%-54%) instead of the acute value threshold (21%). Serum protein concentration and HSA significantly decreased in anaphylaxis (p < .0001). HSA levels dropped with the severity of the reaction (6% and 15% for Grade 2 and 3 reactions, respectively). Furthermore, HBB levels increased during the acute phase of all hypersensitivity reactions (p < .0001). CONCLUSIONS: For the first time, the extravasation underlying human anaphylaxis has been evaluated based on the severity of the reaction using HSA and protein concentration measurements. Additionally, our findings propose new diagnostic and potential therapeutic approaches for this pathological event.


Assuntos
Anafilaxia , Humanos , Anafilaxia/diagnóstico , Anafilaxia/etiologia , Triptases , Albumina Sérica Humana
10.
Arch. pediatr. Urug ; 93(2): e401, dic. 2022. tab
Artigo em Espanhol | LILACS, UY-BNMED, BNUY | ID: biblio-1411580

RESUMO

Desde el año 2007 en Uruguay los cuidados paliativos (CP) son parte de las prestaciones de salud que todos los ciudadanos que los necesitan tienen derecho a recibir y, desde entonces, ha aumentado significativamente la accesibilidad a estos. Objetivo: describir la situación actual del desarrollo organizativo de servicios de cuidados paliativos pediátricos (CPP) en el país y los pacientes por ellos asistidos desde el inicio de sus actividades hasta el 31/12/2020, las principales fortalezas y desafíos percibidos por los profesionales de dichos equipos. Metodología: se realizó una consulta mediante encuesta online auto administrada enviada a los coordinadores de servicios de CPP del Uruguay. Resultados: se confirmaron 19 equipos en 9/19 departamentos, 5/19 están integrados por profesionales de las cuatro disciplinas básicas recomendadas, el resto por distintas combinaciones de disciplinas, con cargas horarias muy variables. Brindan asistencia en: hospitalización 19/19, policlínica 18/19, atención domiciliaria coordinada 13/19 y retén telefónico 10/19. Fueron asistidos 2957 niños, 23% de los mismos fallecieron. 16/19 equipos reportan como principales fortalezas los valores compartidos y el trabajo en equipo interdisciplinario y 15/19 como principal desafío los déficits de recursos humanos. Conclusiones: persisten importantes inequidades en el acceso a los CPP. Se constató gran variabilidad en la integración de los equipos y la carga horaria de los profesionales. Es necesario que las autoridades sanitarias continúen promoviendo y exigiendo el desarrollo de equipos de CPP en las instituciones y departamentos que no los tienen y el cumplimiento de estándares mínimos de calidad en los ya existentes.


Since 2007, palliative care (PC) has been a part of the health benefits that all Uruguayan citizens are entitled to receive and, since then, accessibility has increased significantly. Objective: to describe the present development of pediatric palliative care services (PPC) in Uruguay and the patients assisted by health providers since the beginning of their services until 12/31/2020 and the key strengths and challenges perceived by these palliative care teams. Methodology: a consultation was carried out through a self-administered online survey and sent to the PPC service coordinators in Uruguay. Results: 19 teams were confirmed in 9/19 departments, 5/19 are integrated by professionals from the four recommended basic disciplines, the rest by different combinations of disciplines, with highly variable workloads. They provide assistance in: hospitalization 19/19, clinics 18/19, coordinated home care 13/19 and telephone assistance 10/19. 2957 children were assisted, 23% of them died. 16/19 teams report shared values and interdisciplinary teamwork as their main strengths, and 15/19 report human resource shortage as their main challenge. Conclusions: significant inequality persist regarding access to PPCs. We confirmed a high variability in teams' integration and professional workload. It is necessary for the health authorities to continue to promote and demand the development of PPC teams in the institutions and departments that do not yet have them and the compliance with minimum quality standards in those that already operate.


Desde 2007, os cuidados paliativos (CP) fazem parte dos benefícios de saúde que todos os cidadãos têm direito a receber no Uruguai e, desde então, a acessibilidade a eles tem aumentado significativamente. Objetivo: descrever a situação atual do desenvolvimento organizacional dos serviços de cuidados paliativos pediátricos (CPP) no Uruguai e dos pacientes atendidos desde o início de suas atividades até 31/12/2020 e as principais fortalezas e desafios percebidos pelos profissionais das referidas equipes. Metodologia: foi realizada uma consulta por meio de uma pesquisa online autoaplicável enviada aos coordenadores dos serviços do CPP no Uruguai. Resultados: 19 equipes foram confirmadas em 19/09 departamentos, 19/05 compostas por profissionais das quatro disciplinas básicas recomendadas, o restante por diferentes combinações de disciplinas, com cargas horárias altamente variáveis. Elas atendem em: internação 19/19, policlínica 18/19, atendimento domiciliar coordenado 13/19 e posto telefônico 19/10. 2.957 crianças foram atendidas, 23% delas faleceram. 16/19 equipes relatam valores compartilhados e trabalho em equipe interdisciplinar como suas principais fortalezas, e 15/19 relatam déficits de recursos humanos como seu principal desafio. Conclusões: persistem desigualdades significativas no acesso aos CPP. Verificou-se: grande variabilidade na integração das equipes e na carga de trabalho dos profissionais. É necessário que as autoridades de saúde continuem promovendo e exigindo o desenvolvimento de equipes de CPP nas instituições e departamentos que não as possuem e o cumprimento de padrões mínimos de qualidade nas que já existem.


Assuntos
Humanos , Cuidados Paliativos/estatística & dados numéricos , Pediatria/estatística & dados numéricos , Pessoal de Saúde/estatística & dados numéricos , Acessibilidade aos Serviços de Saúde/estatística & dados numéricos , Cuidados Paliativos/organização & administração , Uruguai , Pesquisas sobre Atenção à Saúde
11.
Curr Opin Struct Biol ; 77: 102463, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36183446

RESUMO

S-acylation is a reversible posttranslational modification, where a long-chain fatty acid is attached to a protein through a thioester linkage. Being the most abundant form of lipidation in humans, a family of twenty-three human zDHHC integral membrane enzymes catalyze this reaction. Previous structures of the apo and lipid bound zDHHCs shed light into the molecular details of the active site and binding pocket. Here, we delve further into the details of fatty acyl-CoA recognition by zDHHC acyltransferases using insights from the recent structure. We additionally review indirect evidence that suggests acyl-CoAs do not diffuse freely in the cytosol, but are channeled into specific pathways, and comment on the suggested mechanisms for fatty acyl-CoA compartmentalization and intracellular transport, to finally speculate about the potential mechanisms that underlie fatty acyl-CoA delivery to zDHHC enzymes.


Assuntos
Acetiltransferases , Acil Coenzima A , Aciltransferases , Humanos , Acetiltransferases/metabolismo , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Acilação , Processamento de Proteína Pós-Traducional , Aciltransferases/química , Aciltransferases/metabolismo
12.
Cell Mol Life Sci ; 79(11): 546, 2022 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-36221013

RESUMO

The majority of current cancer therapies are aimed at reducing tumour growth, but there is lack of viable pharmacological options to reduce the formation of metastasis. This is a paradox, since more than 90% of cancer deaths are attributable to metastatic progression. Integrin alpha9 (ITGA9) has been previously described as playing an essential role in metastasis; however, little is known about the mechanism that links this protein to this process, being one of the less studied integrins. We have now deciphered the importance of ITGA9 in metastasis and provide evidence demonstrating its essentiality for metastatic dissemination in rhabdomyosarcoma and neuroblastoma. However, the most translational advance of this study is to reveal, for the first time, the possibility of reducing metastasis by pharmacological inhibition of ITGA9 with a synthetic peptide simulating a key interaction domain of ADAM proteins, in experimental metastasis models, not only in childhood cancers but also in a breast cancer model.


Assuntos
Neuroblastoma , Rabdomiossarcoma , Proteínas ADAM/metabolismo , Humanos , Cadeias alfa de Integrinas , Integrinas , Metástase Neoplásica , Neuroblastoma/tratamento farmacológico , Rabdomiossarcoma/tratamento farmacológico
13.
Proc Natl Acad Sci U S A ; 119(31): e2202080119, 2022 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-35901214

RESUMO

Protein secretion is an essential process that drives cell growth, movement, and communication. Protein traffic within the secretory pathway occurs via transport intermediates that bud from one compartment and fuse with a downstream compartment to deliver their contents. Here, we explore the possibility that protein secretion can be selectively inhibited by perturbing protein-protein interactions that drive capture into transport vesicles. Human proprotein convertase subtilisin/kexin type 9 (PCSK9) is a determinant of cholesterol metabolism whose secretion is mediated by a specific cargo adaptor protein, SEC24A. We map a series of protein-protein interactions between PCSK9, its endoplasmic reticulum (ER) export receptor SURF4, and SEC24A that mediate secretion of PCSK9. We show that the interaction between SURF4 and SEC24A can be inhibited by 4-phenylbutyrate (4-PBA), a small molecule that occludes a cargo-binding domain of SEC24. This inhibition reduces secretion of PCSK9 and additional SURF4 clients that we identify by mass spectrometry, leaving other secreted cargoes unaffected. We propose that selective small-molecule inhibition of cargo recognition by SEC24 is a potential therapeutic intervention for atherosclerosis and other diseases that are modulated by secreted proteins.


Assuntos
Retículo Endoplasmático , Proteínas de Membrana , Pró-Proteína Convertase 9 , Proteínas de Transporte Vesicular , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Fenilbutiratos , Pró-Proteína Convertase 9/metabolismo , Mapeamento de Interação de Proteínas , Transporte Proteico , Via Secretória , Proteínas de Transporte Vesicular/metabolismo
14.
Int J Mol Sci ; 22(23)2021 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-34884726

RESUMO

The Wnt/ß-catenin signaling pathway plays a pivotal role during embryogenesis and its deregulation is a key mechanism in the origin and progression of several tumors. Wnt antagonists have been described as key modulators of Wnt/ß-catenin signaling in cancer, with Dickkopf-1 (DKK-1) being the most studied member of the DKK family. Although the therapeutic potential of DKK-1 inhibition has been evaluated in several diseases and malignancies, little is known in pediatric tumors. Only a few works have studied the genetic inhibition and function of DKK-1 in rhabdomyosarcoma. Here, for the first time, we report the analysis of the therapeutic potential of DKK-1 pharmaceutical inhibition in rhabdomyosarcoma, the most common soft tissue sarcoma in children. We performed DKK-1 inhibition via shRNA technology and via the chemical inhibitor WAY-2626211. Its inhibition led to ß-catenin activation and the modulation of focal adhesion kinase (FAK), with positive effects on in vitro expression of myogenic markers and a reduction in proliferation and invasion. In addition, WAY-262611 was able to impair survival of tumor cells in vivo. Therefore, DKK-1 could constitute a molecular target, which could lead to novel therapeutic strategies in RMS, especially in those patients with high DKK-1 expression.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Naftalenos/uso terapêutico , Piperidinas/uso terapêutico , Pirimidinas/uso terapêutico , Rabdomiossarcoma/tratamento farmacológico , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Animais , Estudos de Casos e Controles , Linhagem Celular Tumoral , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Camundongos SCID , Terapia de Alvo Molecular , Músculos/metabolismo , Proteína MyoD/metabolismo , Miogenina/metabolismo , Naftalenos/farmacologia , Piperidinas/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/uso terapêutico , Rabdomiossarcoma/etiologia , Rabdomiossarcoma/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Pharmaceuticals (Basel) ; 14(8)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34451907

RESUMO

The Wnt signaling pathway regulates crucial aspects such as cell fate determination, cell polarity and organogenesis during embryonic development. Wnt pathway deregulation is a hallmark of several cancers such as lung, gastric and liver cancer, and has been reported to be altered in others. Despite the general agreement reached by the scientific community on the oncogenic potential of the central components of the pathway, the role of the antagonist proteins remains less clear. Deregulation of the pathway may be caused by overexpression or downregulation of a wide range of antagonist proteins. Although there is growing information related to function and regulation of Dickkopf (DKK) proteins, their pharmacological potential as cancer therapeutics still has not been fully developed. This review provides an update on the role of DKK proteins in cancer and possible potential as therapeutic targets for the treatment of cancer; available compounds in pre-clinical or clinical trials are also reviewed.

16.
J Cell Biol ; 219(11)2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-32997735

RESUMO

Protein secretion is initiated at the endoplasmic reticulum by the COPII coat, which self-assembles to form vesicles. Here, we examine the mechanisms by which a cargo-bound inner coat layer recruits and is organized by an outer scaffolding layer to drive local assembly of a stable structure rigid enough to enforce membrane curvature. An intrinsically disordered region in the outer coat protein, Sec31, drives binding with an inner coat layer via multiple distinct interfaces, including a newly defined charge-based interaction. These interfaces combinatorially reinforce each other, suggesting coat oligomerization is driven by the cumulative effects of multivalent interactions. The Sec31 disordered region could be replaced by evolutionarily distant sequences, suggesting plasticity in the binding interfaces. Such a multimodal assembly platform provides an explanation for how cells build a powerful yet transient scaffold to direct vesicle traffic.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Ligação Proteica , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética
17.
Traffic ; 21(11): 702-711, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32975860

RESUMO

The appropriate delivery of secretory proteins to the correct subcellular destination is an essential cellular process. In the endoplasmic reticulum (ER), secretory proteins are captured into COPII vesicles that generally exclude ER resident proteins and misfolded proteins. We previously characterized a collection of yeast mutants that fail to enforce this sorting stringency and improperly secrete the ER chaperone, Kar2 (Copic et al., Genetics 2009). Here, we used the emp24Δ mutant strain that secretes Kar2 to identify candidate proteins that might regulate ER export, reasoning that loss of regulatory proteins would restore sorting stringency. We find that loss of the deubiquitylation complex Ubp3/Bre5 reverses all of the known phenotypes of the emp24Δ mutant, and similarly reverses Kar2 secretion of many other ER retention mutants. Based on a combination of genetic interactions and live cell imaging, we conclude that Ubp3 and Bre5 modulate COPII coat assembly at ER exit sites. Therefore, we propose that Ubp3/Bre5 influences the rate of vesicle formation from the ER that in turn can impact ER quality control events.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório , Proteínas de Saccharomyces cerevisiae , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Transporte Proteico , Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
J Cell Biol ; 219(7)2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32406500

RESUMO

Accurate maintenance of organelle identity in the secretory pathway relies on retention and retrieval of resident proteins. In the endoplasmic reticulum (ER), secretory proteins are packaged into COPII vesicles that largely exclude ER residents and misfolded proteins by mechanisms that remain unresolved. Here we combined biochemistry and genetics with correlative light and electron microscopy (CLEM) to explore how selectivity is achieved. Our data suggest that vesicle occupancy contributes to ER retention: in the absence of abundant cargo, nonspecific bulk flow increases. We demonstrate that ER leakage is influenced by vesicle size and cargo occupancy: overexpressing an inert cargo protein or reducing vesicle size restores sorting stringency. We propose that cargo recruitment into vesicles creates a crowded lumen that drives selectivity. Retention of ER residents thus derives in part from the biophysical process of cargo enrichment into a constrained spherical membrane-bound carrier.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Saccharomyces cerevisiae/metabolismo , Via Secretória/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Retículo Endoplasmático/genética , Retículo Endoplasmático/ultraestrutura , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Reporter , Complexo de Golgi/genética , Complexo de Golgi/ultraestrutura , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Imagem Óptica , Transporte Proteico , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
19.
Curr Opin Cell Biol ; 65: 96-102, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32408120

RESUMO

Misfolded and mistargeted proteins in the early secretory pathway present significant risks to the cell. A diverse and integrated network of quality control pathways protects the cell from these threats. We focus on the discovery of new mechanisms that contribute to this protective network. Biochemical and structural advances in endoplasmic reticulum targeting fidelity, and in the redistribution of mistargeted substrates are discussed. We further review new discoveries in quality control at the inner nuclear membrane in the context of orphaned subunits. We consider developments in our understanding of cargo selection for endoplasmic reticulum export. Conflicting data on quality control by cargo receptor proteins are discussed and we look to important future questions for the field.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Humanos , Modelos Biológicos , Dobramento de Proteína , Via Secretória
20.
Curr Biol ; 30(5): 854-864.e5, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-31956032

RESUMO

Cells possess multiple mechanisms that protect against the accumulation of toxic aggregation-prone proteins. Here, we identify a pre-emptive pathway that reduces synthesis of membrane proteins that have failed to properly assemble in the endoplasmic reticulum (ER). We show that loss of the ER membrane complex (EMC) or mutation of the Sec61 translocon causes reduced synthesis of misfolded forms of the yeast ABC transporter Yor1. Synthesis defects are rescued by various ribosomal mutations, as well as by reducing cellular ribosome abundance. Genetic and biochemical evidence point to a ribosome-associated quality-control pathway triggered by ribosome collisions when membrane domain insertion and/or folding fails. In support of this model, translation initiation also contributes to synthesis defects, likely by modulating ribosome abundance on the message. Examination of translation efficiency across the yeast membrane proteome revealed that polytopic membrane proteins have relatively low ribosome abundance, providing evidence for translational tuning to balance protein synthesis and folding. We propose that by modulating translation rates of poorly folded proteins, cells can pre-emptively protect themselves from potentially toxic aberrant transmembrane proteins.


Assuntos
Membranas Intracelulares/química , Proteínas de Membrana/química , Dobramento de Proteína , Ribossomos/metabolismo , Saccharomyces cerevisiae/química
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