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1.
Proc Natl Acad Sci U S A ; 121(17): e2317680121, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38635626

RESUMO

The endosomal sorting complex required for transport (ESCRT) machinery constitutes multisubunit protein complexes that play an essential role in membrane remodeling and trafficking. ESCRTs regulate a wide array of cellular processes, including cytokinetic abscission, cargo sorting into multivesicular bodies (MVBs), membrane repair, and autophagy. Given the versatile functionality of ESCRTs, and the intricate organizational structure of the ESCRT machinery, the targeted modulation of distinct ESCRT complexes is considerably challenging. This study presents a pseudonatural product targeting IST1-CHMP1B within the ESCRT-III complexes. The compound specifically disrupts the interaction between IST1 and CHMP1B, thereby inhibiting the formation of IST1-CHMP1B copolymers essential for normal-topology membrane scission events. While the compound has no impact on cytokinesis, MVB sorting, or biogenesis of extracellular vesicles, it rapidly inhibits transferrin receptor recycling in cells, resulting in the accumulation of transferrin in stalled sorting endosomes. Stalled endosomes become decorated by lipidated LC3, suggesting a link between noncanonical LC3 lipidation and inhibition of the IST1-CHMP1B complex.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos , Endossomos/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Transporte Proteico , Corpos Multivesiculares/metabolismo
2.
Autophagy ; 20(2): 443-444, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-37872727

RESUMO

ATG16L1 is an essential component of the Atg8-family protein conjugation machinery, providing membrane targeting for the ATG12-ATG5 conjugate. Recently, we identified an alternative E3-like complex that functions independently of ATG16L1. This complex utilizes the autophagosome-lysosome tethering factor TECPR1 for membrane targeting. TECPR1 is recruited to damaged lysosomal membranes via a direct interaction with sphingomyelin. At the damaged membrane, TECPR1 assembles into an E3-like complex with ATG12-ATG5 to regulate unconventional LC3 lipidation and promote efficient lysosomal repair.


Assuntos
Autofagia , Proteínas Associadas aos Microtúbulos , Proteína 5 Relacionada à Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína 12 Relacionada à Autofagia , Lisossomos/metabolismo , Proteínas Relacionadas à Autofagia
3.
EMBO Rep ; 24(9): e56841, 2023 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-37381828

RESUMO

Lysosomal membrane damage represents a threat to cell viability. As such, cells have evolved sophisticated mechanisms to maintain lysosomal integrity. Small membrane lesions are detected and repaired by the endosomal sorting complex required for transport (ESCRT) machinery while more extensively damaged lysosomes are cleared by a galectin-dependent selective macroautophagic pathway (lysophagy). In this study, we identify a novel role for the autophagosome-lysosome tethering factor, TECPR1, in lysosomal membrane repair. Lysosomal damage promotes TECPR1 recruitment to damaged membranes via its N-terminal dysferlin domain. This recruitment occurs upstream of galectin and precedes the induction of lysophagy. At the damaged membrane, TECPR1 forms an alternative E3-like conjugation complex with the ATG12-ATG5 conjugate to regulate ATG16L1-independent unconventional LC3 lipidation. Abolishment of LC3 lipidation via ATG16L1/TECPR1 double knockout impairs lysosomal recovery following damage.


Assuntos
Autofagia , Proteínas Associadas aos Microtúbulos , Proteínas Associadas aos Microtúbulos/metabolismo , Macroautofagia , Galectinas/metabolismo , Lisossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo
4.
Autophagy ; 19(6): 1885-1886, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36409136

RESUMO

Extracellular pathogens utilize secreted virulence factors to regulate host cell function. Recently we characterized the molecular mechanism behind host macroautophagy/autophagy regulation by the Vibrio cholerae toxin MakA. Cholesterol binding at the plasma membrane induces MakA endocytosis and pH-dependent pore assembly. Membrane perforation of late endosomal membranes induces cellular membrane repair pathways and V-ATPase-dependent unconventional LC3 lipidation on damaged membranes.


Assuntos
Autofagia , Vibrio cholerae , Vibrio cholerae/química , Vibrio cholerae/metabolismo , Membrana Celular/metabolismo , Fagocitose , Fatores de Virulência/metabolismo , Proteínas de Bactérias/metabolismo
5.
J Cell Biol ; 221(12)2022 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-36194176

RESUMO

Pore-forming toxins (PFTs) are important virulence factors produced by many pathogenic bacteria. Here, we show that the Vibrio cholerae toxin MakA is a novel cholesterol-binding PFT that induces non-canonical autophagy in a pH-dependent manner. MakA specifically binds to cholesterol on the membrane at pH < 7. Cholesterol-binding leads to oligomerization of MakA on the membrane and pore formation at pH 5.5. Unlike other cholesterol-dependent cytolysins (CDCs) which bind cholesterol through a conserved cholesterol-binding motif (Thr-Leu pair), MakA contains an Ile-Ile pair that is essential for MakA-cholesterol interaction. Following internalization, endosomal acidification triggers MakA pore-assembly followed by ESCRT-mediated membrane repair and V-ATPase-dependent unconventional LC3 lipidation on the damaged endolysosomal membranes. These findings characterize a new cholesterol-binding toxin that forms pores in a pH-dependent manner and reveals the molecular mechanism of host autophagy manipulation.


Assuntos
Autofagia , Proteínas de Bactérias , Colesterol , Citotoxinas , Vibrio cholerae , Fatores de Virulência , Adenosina Trifosfatases/metabolismo , Motivos de Aminoácidos , Autofagia/efeitos dos fármacos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Colesterol/metabolismo , Citotoxinas/metabolismo , Citotoxinas/farmacologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/química , Endossomos/metabolismo , Concentração de Íons de Hidrogênio , Lisossomos/química , Lisossomos/metabolismo , Ligação Proteica , Vibrio cholerae/química , Vibrio cholerae/metabolismo , Fatores de Virulência/química , Fatores de Virulência/metabolismo
6.
Chem Sci ; 13(24): 7240-7246, 2022 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-35799822

RESUMO

Chemical modification of proteins is enormously useful for characterizing protein function in complex biological systems and for drug development. Selective labeling of native or endogenous proteins is challenging owing to the existence of distinct functional groups in proteins and in living systems. Chemistry for rapid and selective labeling of proteins remains in high demand. Here we have developed novel affinity labeling probes using benzotriazole (BTA) chemistry. We showed that affinity-based BTA probes selectively and covalently label a lysine residue in the vicinity of the ligand binding site of a target protein with a reaction half-time of 28 s. The reaction rate constant is comparable to the fastest biorthogonal chemistry. This approach was used to selectively label different cytosolic and membrane proteins in vitro and in live cells. BTA chemistry could be widely useful for labeling of native/endogenous proteins, target identification and development of covalent inhibitors.

7.
J Cell Sci ; 134(5)2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33106317

RESUMO

Autophagy plays an essential role in the defense against many microbial pathogens as a regulator of both innate and adaptive immunity. Some pathogens have evolved sophisticated mechanisms that promote their ability to evade or subvert host autophagy. Here, we describe a novel mechanism of autophagy modulation mediated by the recently discovered Vibrio cholerae cytotoxin, motility-associated killing factor A (MakA). pH-dependent endocytosis of MakA by host cells resulted in the formation of a cholesterol-rich endolysosomal membrane aggregate in the perinuclear region. Aggregate formation induced the noncanonical autophagy pathway driving unconventional LC3 (herein referring to MAP1LC3B) lipidation on endolysosomal membranes. Subsequent sequestration of the ATG12-ATG5-ATG16L1 E3-like enzyme complex, required for LC3 lipidation at the membranous aggregate, resulted in an inhibition of both canonical autophagy and autophagy-related processes, including the unconventional secretion of interleukin-1ß (IL-1ß). These findings identify a novel mechanism of host autophagy modulation and immune modulation employed by V. cholerae during bacterial infection.


Assuntos
Proteínas Associadas aos Microtúbulos , Vibrio cholerae , Autofagia , Proteínas Relacionadas à Autofagia/genética , Citotoxinas , Vitamina B 12/análogos & derivados
8.
Nat Chem Biol ; 15(7): 710-720, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31222192

RESUMO

Autophagy mediates the degradation of damaged proteins, organelles and pathogens, and plays a key role in health and disease. Thus, the identification of new mechanisms involved in the regulation of autophagy is of major interest. In particular, little is known about the role of lipids and lipid-binding proteins in the early steps of autophagosome biogenesis. Using target-agnostic, high-content, image-based identification of indicative phenotypic changes induced by small molecules, we have identified autogramins as a new class of autophagy inhibitor. Autogramins selectively target the recently discovered cholesterol transfer protein GRAM domain-containing protein 1A (GRAMD1A, which had not previously been implicated in autophagy), and directly compete with cholesterol binding to the GRAMD1A StART domain. GRAMD1A accumulates at sites of autophagosome initiation, affects cholesterol distribution in response to starvation and is required for autophagosome biogenesis. These findings identify a new biological function of GRAMD1A and a new role for cholesterol in autophagy.


Assuntos
Autofagossomos/metabolismo , Proteínas de Membrana/metabolismo , Autofagossomos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Humanos , Proteínas de Membrana/antagonistas & inibidores , Modelos Moleculares , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Células Tumorais Cultivadas
9.
Exp Cell Res ; 340(1): 125-31, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26712520

RESUMO

The pre-mRNA splicing factor 4 kinase PRP4K (PRPF4B), is an essential kinase that is a component of the U5 snRNP and functions in spliceosome assembly. We demonstrated that PRP4K is a novel biological marker for taxane response in ovarian cancer patients and reduced levels of PRP4K correlate with intrinsic and acquired taxane resistance in both breast and ovarian cancer. Breast cancer treatments are chosen based on hormone and growth factor receptor status, with HER2 (ERBB2) positive breast cancer patients receiving anti-HER2 agents and taxanes and estrogen receptor alpha (ESR1) positive (ER+) breast cancer patients receiving anti-estrogen therapies such as tamoxifen. Here we demonstrate that PRP4K is expressed in the normal mammary duct epithelial cells of the mouse, and that estrogen induces PRP4K gene and protein expression in ER+ human MCF7 breast cancer cells. Estrogen acts through ESR1 to regulate PRP4K expression, as over-expression of ESR1 in the ER-negative MDA-MB-231 breast cancer cell line increased the expression of this kinase, and knock-down of ESR1 in ER+ T47D breast cancer cells reduced PRP4K levels. Furthermore, treatment with 4-hydroxytamoxifen (4-OHT) resulted in a dose-dependent decrease in PRP4K protein expression in MCF7 cells. Consistent with our previous studies identifying PRP4K as a taxane-response biomarker, reduced PRP4K expression in 4-OHT-treated cells correlated with reduced sensitivity to paclitaxel. Thus, PRP4K is novel estrogen regulated kinase, and its levels can be reduced by 4-OHT in ER+ breast cancer cells altering their response to taxanes.


Assuntos
Biomarcadores Tumorais/genética , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Receptor alfa de Estrogênio/metabolismo , Proteínas Serina-Treonina Quinases/genética , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Transdução de Sinais , Taxoides/farmacologia , Animais , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Camundongos , Paclitaxel/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/antagonistas & inibidores , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Relação Estrutura-Atividade , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Células Tumorais Cultivadas
10.
Nucleus ; 6(4): 279-88, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26098145

RESUMO

Alternative pre-mRNA splicing in higher eukaryotes enhances transcriptome complexity and proteome diversity. Its regulation is mediated by a complex RNA-protein network that is essential for the maintenance of cellular and tissue homeostasis. Disruptions to this regulatory network underlie a host of human diseases and contribute to cancer development and progression. The splicing kinases are an important family of pre-mRNA splicing regulators, , which includes the CDC-like kinases (CLKs), the SRSF protein kinases (SRPKs) and pre-mRNA splicing 4 kinase (PRP4K/PRPF4B). These splicing kinases regulate pre-mRNA splicing via phosphorylation of spliceosomal components and serine-arginine (SR) proteins, affecting both their nuclear localization within nuclear speckle domains as well as their nucleo-cytoplasmic shuttling. Here we summarize the emerging evidence that splicing kinases are dysregulated in cancer and play important roles in both tumorigenesis as well as therapeutic response to radiation and chemotherapy.


Assuntos
Processamento Alternativo , Carcinogênese , Proteínas Serina-Treonina Quinases/genética , Arginina/metabolismo , Linhagem Celular Tumoral , Redes Reguladoras de Genes , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Serina/metabolismo
11.
Cancer Cell ; 27(5): 682-97, 2015 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-25965573

RESUMO

Metastatic dissemination is the leading cause of death in cancer patients, which is particularly evident for high-risk sarcomas such as Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma. Previous research identified a crucial role for YB-1 in the epithelial-to-mesenchymal transition (EMT) and metastasis of epithelial malignancies. Based on clinical data and two distinct animal models, we now report that YB-1 is also a major metastatic driver in high-risk sarcomas. Our data establish YB-1 as a critical regulator of hypoxia-inducible factor 1α (HIF1α) expression in sarcoma cells. YB-1 enhances HIF1α protein expression by directly binding to and activating translation of HIF1A messages. This leads to HIF1α-mediated sarcoma cell invasion and enhanced metastatic capacity in vivo, highlighting a translationally regulated YB-1-HIF1α axis in sarcoma metastasis.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Metástase Neoplásica , Biossíntese de Proteínas , Sarcoma/patologia , Proteína 1 de Ligação a Y-Box/fisiologia , Humanos , Invasividade Neoplásica , Sarcoma/genética , Proteína Supressora de Tumor Von Hippel-Lindau/fisiologia
12.
Cell Cycle ; 14(7): 1059-69, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25602630

RESUMO

The taxanes are used alone or in combination with anthracyclines or platinum drugs to treat breast and ovarian cancer, respectively. Taxanes target microtubules in cancer cells and modifiers of taxane sensitivity have been identified in vitro, including drug efflux and mitotic checkpoint proteins. Human epidermal growth factor receptor 2 (HER2/ERBB2) gene amplification is associated with benefit from taxane therapy in breast cancer yet high HER2 expression also correlates with poor survival in both breast and ovarian cancer. The pre-mRNA splicing factor 4 kinase PRP4K (PRPF4B), which we identified as a component of the U5 snRNP also plays a role in regulating the spindle assembly checkpoint (SAC) in response to microtubule-targeting drugs. In this study, we found a positive correlation between PRP4K expression and HER2 status in breast and ovarian cancer patient tumors, which we determined was a direct result of PRP4K regulation by HER2 signaling. Knock-down of PRP4K expression reduced the sensitivity of breast and ovarian cancer cell lines to taxanes, and low PRP4K levels correlated with in vitro-derived and patient acquired taxane resistance in breast and ovarian cancer. Patients with high-grade serous ovarian cancer and high HER2 levels had poor overall survival; however, better survival in the low HER2 patient subgroup treated with platinum/taxane-based therapy correlated positively with PRP4K expression (HR = 0.37 [95% CI 0.15-0.88]; p = 0.03). Thus, PRP4K functions as a HER2-regulated modifier of taxane sensitivity that may have prognostic value as a marker of better overall survival in taxane-treated ovarian cancer patients.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/enzimologia , Neoplasias Ovarianas/enzimologia , Paclitaxel/farmacologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptor ErbB-2/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/fisiologia , Neoplasias da Mama/mortalidade , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Amplificação de Genes , Humanos , Estimativa de Kaplan-Meier , Células MCF-7 , Neoplasias Ovarianas/mortalidade , Receptor ErbB-2/genética , Transdução de Sinais
13.
Haematologica ; 100(1): 70-6, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25281505

RESUMO

Cancer therapeutics is evolving to precision medicine, with the goal of matching targeted compounds with molecular aberrations underlying a patient's cancer. While murine models offer a pre-clinical tool, associated costs and time are not compatible with actionable patient-directed interventions. Using the paradigm of T-cell acute lymphoblastic leukemia, a high-risk disease with defined molecular underpinnings, we developed a zebrafish human cancer xenotransplantation model to inform therapeutic decisions. Using a focused chemical genomic approach, we demonstrate that xenografted cell lines harboring mutations in the NOTCH1 and PI3K/AKT pathways respond concordantly to their targeted therapies, patient-derived T-cell acute lymphoblastic leukemia can be successfully engrafted in zebrafish and specific drug responses can be quantitatively determined. Using this approach, we identified a mutation sensitive to γ-secretase inhibition in a xenograft from a child with T-cell acute lymphoblastic leukemia, confirmed by Sanger sequencing and validated as a gain-of-function NOTCH1 mutation. The zebrafish xenotransplantation platform provides a novel cost-effective means of tailoring leukemia therapy in real time.


Assuntos
Antineoplásicos/farmacologia , Embrião não Mamífero/efeitos dos fármacos , Genômica/métodos , Mutação/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Peixe-Zebra/genética , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Células Cultivadas , Criança , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Inibidores Enzimáticos/farmacologia , Imunofluorescência , Células HeLa , Humanos , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Receptor Notch1/genética , Transdução de Sinais , Transplante Heterólogo , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo
14.
Bioorg Med Chem ; 21(19): 5995-6002, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23958515

RESUMO

To generate the first series of prodigiosene conjugates, the tripyrrolic skeleton was appended to estrone, tamoxifen and porphyrin frameworks by way of ester linkers and various hydrocarbon chain lengths. The ability of the conjugates to inhibit various types of cancer cells was evaluated in vitro. The porphyrin conjugates did not exhibit significant activity. The estrone conjugates exhibited modest activity, for the most part. However, significantly greater growth inhibition activity against certain breast, colon, lung, leukemia, melanoma and prostate cell lines was noted. This unusual effect for this first generation model class of compound warrants further investigation and comparison to cases where estrogens are linked to prodigiosenes via connection points that do not feature in estrogen receptor binding. The 4-hydroxytamoxifen conjugates exhibit nanomolar range activity against the MCF-7 breast cancer cell line, paving the way to expand the scope and connectivity of prodigiosene-tamoxifen conjugates.


Assuntos
Estrona/síntese química , Neoplasias/tratamento farmacológico , Porfirinas/química , Prodigiosina/química , Tamoxifeno/análogos & derivados , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Estrona/química , Estrona/farmacologia , Humanos , Células MCF-7 , Estrutura Molecular , Tamoxifeno/química
15.
Org Biomol Chem ; 11(1): 62-8, 2013 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-23070266

RESUMO

Prodigiosenes, possessing a 4-methoxypyrrolyldipyrrin skeleton, are known for their anti-cancer activity. Structural modification of the C-ring resulted in a series of prodigiosenes that displayed promising activity against leukemia cell lines during in vitro analysis against the NCI 60 cancer cell line panel. Further in vivo studies of these compounds using the zebrafish model showed persistence of anti-leukemia properties in human K562 chronic myelogenous leukemia cells.


Assuntos
Antineoplásicos/farmacologia , Leucemia/tratamento farmacológico , Neoplasias Experimentais/tratamento farmacológico , Prodigiosina/farmacologia , Pirróis/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Leucemia/patologia , Estrutura Molecular , Neoplasias Experimentais/patologia , Prodigiosina/análogos & derivados , Prodigiosina/química , Pirróis/síntese química , Pirróis/química , Relação Estrutura-Atividade , Peixe-Zebra
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