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1.
Neurooncol Adv ; 5(1): vdad028, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37128507

RESUMO

Background: Tumor surveillance of isocitrate dehydrogenase (IDH) mutant gliomas is accomplished via serial contrast MRI. When new contrast enhancement (CEnew) is detected during postsurgical surveillance, clinicians must assess whether CEnew indicates pseudoprogression (PsP) or tumor progression (TP). PsP has been better studied in IDH wild-type glioblastoma but has not been well characterized in IDH mutant gliomas. We conducted a retrospective study evaluating the incidence, predictors, natural history, and survival of PsP patients in a large cohort of IDH mutant glioma patients treated at a single institution. Methods: We identified 587 IDH mutant glioma patients treated at UCLA. We directly inspected MRI images and radiology reports to identify CEnew and categorized CEnew into TP or PsP using MRI or histopathology. Results: Fifty-six percent of patients developed CEnew (326/587); of these, 92/326 patients (28% of CEnew; 16% of all) developed PsP and 179/326 (55%) developed TP. All PsP patients had prior radiation, chemotherapy, or chemoradiotherapy. PsP was associated with longer overall survival (OS) versus TP patients and similar OS versus no CEnew. PsP differs from TP based on earlier time of onset (median 5.8 vs 17.4 months from treatment, P < .0001) and MRI features that include punctate enhancement and enhancement location. Conclusion: PsP patients represented 28% of CEnew patients and 16% of all patients; PsP patients demonstrated superior outcomes to TP patients, and equivalent survival to patients without CEnew. PsP persists for <1 year, occurs after treatment, and differs from TP based on time of onset and radiographic features. Poor outcomes after CEnew are driven by TP.

2.
J Biol Chem ; 296: 100230, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33361156

RESUMO

Post-translational modification of protein by ubiquitin (Ub) alters the stability, subcellular location, or function of the target protein, thereby impacting numerous biological processes and directly contributing to myriad cellular defects or disease states, such as cancer. Tracking substrate ubiquitination by fluorescence provides opportunities for advanced reaction dynamics studies and for translational research including drug discovery. However, fluorescence-based techniques in ubiquitination studies remain underexplored at least partly because of challenges associated with Ub chain complexity and requirement for additional substrate modification. Here we describe a general strategy, FRET diubiquitination, to track substrate ubiquitination by fluorescence. This platform produces a uniform di-Ub product depending on specific interactions between a substrate and its cognate E3 Ub ligase. The diubiquitination creates proximity between the Ub-linked donor and acceptor fluorophores, respectively, enabling energy transfer to yield a distinct fluorescent signal. FRET diubiquitination relies on Ub-substrate fusion, which can be implemented using either one of the two validated strategies. Method 1 is the use of recombinant substrate-Ub fusion, applicable to all substrate peptides that can bind to E3. Method 2 is a chemoenzymatic ligation approach that employs synthetic chemistry to fuse Ub with a substrate peptide containing desired modification. Taken together, our new FRET-based diubiquitination system provides a timely technology of potential to advance both basic research and translation sciences.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Inibidor de NF-kappaB alfa/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , beta Catenina/metabolismo , Sequência de Aminoácidos , Corantes Fluorescentes/química , Humanos , Inibidor de NF-kappaB alfa/genética , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Células-Tronco/genética , Fator de Células-Tronco/metabolismo , Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , beta Catenina/genética
3.
Acta Neurol Scand ; 139(1): 82-85, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30216413

RESUMO

BACKGROUND: To assess the utility of Magnetic Resonance Spectroscopy (MRS) as a biomarker of response to L-arginine in mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS). AIMS: To describe a case of MELAS treated with L-arginine that showed improvement clinically and on serial MRS METHODS: MRS was performed on a 1.5-Tesla scanner to evaluate a MELAS patient before, during, and after intravenous (IV) L-arginine therapy for the treatment of stroke-like episodes. L-arginine was infused at a dose of 500 mg/kg daily for 7 days followed by oral arginine therapy. RESULTS: The patient had clinical improvement after treatment with IV L-arginine. MRS performed before, during, and after treatment with IV L-arginine showed significant improvement in brain lactate and increase in the N-acetylaspartate/Choline (NAA/Cho) ratio compared to pre-treatment baseline. CONCLUSION: Serial MRS imaging showed significant improvement in lactate peaks and NAA/Cho ratios that corresponded with clinical improvement after L-arginine therapy. Given this correlation between radiologic and clinical improvement, MRS may be a useful biomarker assessing response to treatment in MELAS.


Assuntos
Encéfalo/diagnóstico por imagem , Síndrome MELAS/diagnóstico por imagem , Síndrome MELAS/tratamento farmacológico , Espectroscopia de Ressonância Magnética/métodos , Arginina/uso terapêutico , Ácido Aspártico/análogos & derivados , Ácido Aspártico/análise , Colina/análise , Feminino , Humanos , Síndrome MELAS/complicações , Pessoa de Meia-Idade , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/etiologia , Resultado do Tratamento
4.
Proc Natl Acad Sci U S A ; 113(14): E2011-8, 2016 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-27001857

RESUMO

Cullin-RING E3 ubiquitin ligases (CRL) control a myriad of biological processes by directing numerous protein substrates for proteasomal degradation. Key to CRL activity is the recruitment of the E2 ubiquitin-conjugating enzyme Cdc34 through electrostatic interactions between E3's cullin conserved basic canyon and the acidic C terminus of the E2 enzyme. This report demonstrates that a small-molecule compound, suramin, can inhibit CRL activity by disrupting its ability to recruit Cdc34. Suramin, an antitrypansomal drug that also possesses antitumor activity, was identified here through a fluorescence-based high-throughput screen as an inhibitor of ubiquitination. Suramin was shown to target cullin 1's conserved basic canyon and to block its binding to Cdc34. Suramin inhibits the activity of a variety of CRL complexes containing cullin 2, 3, and 4A. When introduced into cells, suramin induced accumulation of CRL substrates. These observations help develop a strategy of regulating ubiquitination by targeting an E2-E3 interface through small-molecule modulators.


Assuntos
Ligases/antagonistas & inibidores , Suramina/farmacologia , Relação Estrutura-Atividade
5.
Methods Mol Biol ; 1280: 339-54, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25736759

RESUMO

A key regulatory node in NF-κB signaling is the removal of the IκBα inhibitor, whose levels are tightly controlled by the ubiquitin-proteasome system. In response to signal activation and transmission, ubiquitin E1, E2, and E3 enzymes are employed to generate a lysine 48-linked ubiquitin chain that triggers degradation of IκBα by the proteasome. In this chapter we describe an in vitro biochemical approach to reconstitute the ubiquitination system. To do so, we detail methods for the preparation of the relevant enzymes and substrate, as well as for the execution of the reaction with high efficiency. This sensitive and highly reproducible readout can be applied to the study of proteins, small molecules, and other factors that modulate IκBα ubiquitination, thereby producing outcomes that impact NF-κB signaling to advance the course of improving human health.


Assuntos
Proteínas I-kappa B/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitinação , Linhagem Celular , Humanos , Técnicas In Vitro , Inibidor de NF-kappaB alfa , Proteólise , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo
6.
Proc Natl Acad Sci U S A ; 111(23): 8434-9, 2014 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-24912152

RESUMO

Lysine 48 (K48)-polyubiquitination is the predominant mechanism for mediating selective protein degradation, but the underlying molecular basis of selecting ubiquitin (Ub) K48 for linkage-specific chain synthesis remains elusive. Here, we present biochemical, structural, and cell-based evidence demonstrating a pivotal role for the Ub Y59-E51 loop in supporting K48-polyubiquitination. This loop is established by a hydrogen bond between Ub Y59's hydroxyl group and the backbone amide of Ub E51, as substantiated by NMR spectroscopic analysis. Loop residues Y59 and R54 are specifically required for the receptor activity enabling K48 to attack the donor Ub-E2 thiol ester in reconstituted ubiquitination catalyzed by Skp1-Cullin1-F-box (SCF)(ßTrCP) E3 ligase and Cdc34 E2-conjugating enzyme. When introduced into mammalian cells, loop-disruptive mutant Ub(R54A/Y59A) diminished the production of K48-polyubiquitin chains. Importantly, conditional replacement of human endogenous Ub by Ub(R54A/Y59A) or Ub(K48R) yielded profound apoptosis at a similar extent, underscoring the global impact of the Ub Y59-E51 loop in cellular K48-polyubiquitination. Finally, disulfide cross-linking revealed interactions between the donor Ub-bound Cdc34 acidic loop and the Ub K48 site, as well as residues within the Y59-E51 loop, suggesting a mechanism in which the Ub Y59-E51 loop helps recruit the E2 acidic loop that aligns the receptor Ub K48 to the donor Ub for catalysis.


Assuntos
Lisina/metabolismo , Poliubiquitina/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Sequência de Aminoácidos , Substituição de Aminoácidos , Apoptose/genética , Biocatálise , Linhagem Celular Tumoral , Células HEK293 , Humanos , Ligação de Hidrogênio , Immunoblotting , Lisina/química , Lisina/genética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Poliubiquitina/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Interferência de RNA , Proteínas Ligases SKP Culina F-Box/química , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitina/química , Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo
7.
J Biol Chem ; 289(10): 7068-7081, 2014 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-24464578

RESUMO

We have explored the mechanisms of polyubiquitin chain assembly with reconstituted ubiquitination of IκBα and ß-catenin by the Skp1-cullin 1-ßTrCP F-box protein (SCF(ßTrCP)) E3 ubiquitin (Ub) ligase complex. Competition experiments revealed that SCF(ßTrCP) formed a complex with IκBα and that the Nedd8 modified E3-substrate platform engaged in dynamic interactions with the Cdc34 E2 Ub conjugating enzyme for chain elongation. Using "elongation intermediates" containing ß-catenin linked with Ub chains of defined length, it was observed that a Lys-48-Ub chain of a length greater than four, but not its Lys-63 linkage counterparts, slowed the rate of additional Ub conjugation. Thus, the Ub chain length and linkage impact kinetic rates of chain elongation. Given that Lys-48-tetra-Ub is packed into compact conformations due to extensive intrachain interactions between Ub subunits, this topology may limit the accessibility of SCF(ßTrCP)/Cdc34 to the distal Ub Lys-48 and result in slowed elongation.


Assuntos
Lisina/metabolismo , Elongação Traducional da Cadeia Peptídica , Poliubiquitina/biossíntese , Ubiquitinação , Células HEK293 , Humanos , Proteínas I-kappa B/metabolismo , Lisina/genética , Proteína NEDD8 , Inibidor de NF-kappaB alfa , Poliubiquitina/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitinas/metabolismo , beta Catenina/metabolismo
8.
J Biol Chem ; 287(40): 33533-44, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-22875853

RESUMO

The application of functional genomic analysis of breast cancer metastasis has led to the identification of a growing number of organ-specific metastasis genes, which often function in concert to facilitate different steps of the metastatic cascade. However, the gene regulatory network that controls the expression of these metastasis genes remains largely unknown. Here, we demonstrate a computational approach for the deconvolution of transcriptional networks to discover master regulators of breast cancer bone metastasis. Several known regulators of breast cancer bone metastasis such as Smad4 and HIF1 were identified in our analysis. Experimental validation of the networks revealed BACH1, a basic leucine zipper transcription factor, as the common regulator of several functional metastasis genes, including MMP1 and CXCR4. Ectopic expression of BACH1 enhanced the malignance of breast cancer cells, and conversely, BACH1 knockdown significantly reduced bone metastasis. The expression of BACH1 and its target genes was linked to the higher risk of breast cancer recurrence in patients. This study established BACH1 as the master regulator of breast cancer bone metastasis and provided a paradigm to identify molecular determinants in complex pathological processes.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Neoplasias Ósseas/secundário , Neoplasias da Mama/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/fisiologia , Regulação Neoplásica da Expressão Gênica , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Osso e Ossos/patologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Feminino , Redes Reguladoras de Genes , Humanos , Hipóxia , Neoplasias Mamárias Animais/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/patologia , Regiões Promotoras Genéticas , Proteínas Smad/metabolismo , Transcrição Gênica
9.
Cancer Cell ; 15(1): 9-20, 2009 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-19111877

RESUMO

Targeted therapy for metastatic diseases relies on the identification of functionally important metastasis genes from a large number of random genetic alterations. Here we use a computational algorithm to map minimal recurrent genomic alterations associated with poor-prognosis breast cancer. 8q22 genomic gain was identified by this approach and validated in an extensive collection of breast tumor samples. Regional gain of 8q22 elevates expression of the metastasis gene metadherin (MTDH), which is overexpressed in more than 40% of breast cancers and is associated with poor clinical outcomes. Functional characterization of MTDH revealed its dual role in promoting metastatic seeding and enhancing chemoresistance. These findings establish MTDH as an important therapeutic target for simultaneously enhancing chemotherapy efficacy and reducing metastasis risk.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Moléculas de Adesão Celular/metabolismo , Cromossomos Humanos Par 8/genética , Resistencia a Medicamentos Antineoplásicos , Genoma Humano/genética , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Humanos , Proteínas de Membrana , Camundongos , Camundongos Nus , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Prognóstico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-met , Proteínas de Ligação a RNA , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/metabolismo , Taxa de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
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